JP2008529035A - Ultra-sensitive detection system using multidimensional signals - Google Patents

Abstract

Disclosed are configurations and methods for sensitive detection of one or more analytes. In general, these methods involve the use of special label components called multidimensional signals. In the disclosed method, as a result of the analysis of the multidimensional signal, another level of analysis can or should be performed, and / or what part of the analyzed material is at another level of analysis. One or more predetermined patterns are generated that can be analyzed or serve to indicate whether or not to analyze. In some formats, isobaric and non-isobaric elements can be used together in the same assay or assay system. Isobaric and non-isobaric multidimensional signals used together can generate one or more predetermined patterns during analysis. The pattern generated in this first level analysis indicates whether a second level analysis should be performed. The second level of analysis may include discrimination of isobaric multidimensional signals.

Description

(Cross-reference of related applications)
This application claims priority to US Provisional Application No. 60 / 649,897, filed February 3, 2005, the entire contents of which are hereby incorporated by reference.

  The present invention relates generally to the field of analyte and biomolecule detection, and in particular to the field of multiplex detection and analysis of analytes and biomolecules.

  Molecular detection is an important task in biomedicine. Such detection often requires the use of specially labeled molecules, signal amplification, or both, since many of the molecules are present in small amounts and do not produce detectable signals themselves. A number of labels, labeling systems, and signal amplification techniques have been developed. For example, the protein may be a sandwich assay (Mailini and Maysef, “Sandwich method of enzyme immunoassay. I. Application to rat and human alphafetoprotein” J. Immunol. Method 8: 223-234 (1975)) and enzyme-linked immunosorbents. Solvent assay (Engvall and Perlmann, “Enzyme-linked immunosorbent assay” (ELISA). Immunoglobulin quantitative assay ”immunochemistry 8: 871-874 (1971)) and two-dimensional (2-D) gel electrophoresis (Patton) , Biotechnology 28: 944-957 (2000)). While these techniques are useful, most have significant drawbacks and limitations. For example, radioactive labels are dangerous and difficult to handle, and fluorescent labels limit the ability to multiplex detection due to the limitations of distinguishable labels. The amplification method tends to be pseudo signal amplification. There is a need for improved detection labels and detection techniques that detect minute amounts of specific molecules and are highly multiplexed.

  Proteome profiling, ie analysis of the expression and presence of proteins such as proteomics, requires sensitive detection of multiple proteins. As current methods in proteome profiling suggest, the tools necessary for such detection are lacking (Haynes and Yates, “Proteome Profiling—Pitfalls and Advances”, Yeast 17 (2): 81-87 (2000)). While chromatographic and capillary electrophoresis techniques are susceptible to proteomic research and have been under considerable development effort (eg, Kulll et al., “Proteins, Nucleic Acids, Peptide Mapping, Biopolymers such as Antibodies, Specific Applications of Capillary Electrochromatography ", J Chromatogr A, 887: 137-63 (2000), Hage," Affinity Chromatography: Clinical Application Summary ", Clin Chem, 45 (5): 593-615 (1999), Hage et al., “chromatography immunoassay”, Anal Chem, 73 (07): 198A-205A, (2001), Krull et al., “Labeling reaction applicable to chromatography and electrophoresis of trace proteins”, J Chromatogr B Biomed Sci Appl, 699: 173-208 (1997)), the industry's mainstay is still two-dimensional electrophoresis in which the two dimensions are isoelectric focus and molecular size. Haynes and Yates point out a significant drawback of the technology, but also discuss the usefulness of the method in view of the drawback. Hayes and Yates describe isotope-coded affinity tag (ICAT) technology, LC-LC-MS / MS technology, and stable isotope labeling technology (Shevchenko et al., “Nanoelectrospray, Isotope Labeling, Quadrupole / Flight. Time A fast “novel” peptide that is sequenced by a combination of mass spectrometers Rapid Commun Mass Mass 11 (9): 1015-1024 (1997); Oda et al., “Accurate protein expression and site-specific phosphorylation. Weighing ", Proc Natl Acad Sci USA 96 (12): 6591-6596 (1999)).

  Abersold et al. (WO 00/11208) describes the labeling of the PRG-LA composition. However, PRG is a protein reactive group, L is a linker (may include an isotopically distinguishable composition), and A is an affinity component. Abersold et al. The protein reactive group is used to label the protein, the affinity capture molecule is used to capture the affinity component, the remaining protein is discarded, the affinity component is released, and the labeled protein is Describes the method detected by mass spectrometry. Abersold et al. This method does not involve labeling or protein fragmentation or other modifications.

  The technique of ICAT, in which cysteine residues are labeled in control and test samples with heavy or light tags, each containing an affinity component, is of great interest and has the potential for protein profiling (Gygi et al. "Quantitative analysis of complex protein mixtures using isotope-encoded affinity tags", Nat. Biotechnol. 17 (10): 994-999 (1999), Griffin et al., "MALDI quadrupole time-of-flight mass spectrometer. Quantitative proteomic analysis used ", Anal. Chem., 73: 978-986 (2001)). Gygi et al. And Griffin et al. Demonstrates the relative profiling of two protein samples, where the two samples are distinguished using a linker containing 8 normal hydrogen or 8 deuterium atoms. The relative concentration of labeled protein is determined by the peak ratio separated by the corresponding 8 amu difference in the linker molecule. Current practice is limited to two labels. This technique does not involve labeling or protein fragmentation or other modifications.

Mass spectrometry has been used to detect phosphorylated proteins (DeGnore and Qin, “Phosphopeptide Fragmentation in Ion Trap Mass Spectrometer”, J. Am. Soc. Mass Spectrom. 9: 1175-1188. (1998); Qin and Chait, “Identification and characterization of post-transcriptional modifications of proteins by MALDI ion trap mass spectrometry”, Anal Chem, 69: 4002-9 (1997); Annan et al., “Multiple Phosphopeptide Mappings”. Dimensional electrospray MS-based approach ", Anal. Chem. 73: 393-404 (2001)). These methods utilize signature masses indicating the presence of phosphate groups, for example m / z = 63 and / or m / z = 79 corresponding to PO ₂ ⁻ and PO ₃ ⁻ ions in the negative ion mode. Alternatively, a neutral loss of 98 daltons from the parent ion indicates a loss of H ₃ PO ₄ from the phosphorylated peptide, phosphorylated Ser, Tyr, Thr. Once the phosphorylated amino acid is identified, the peptide containing the modification is sequenced by standard MS / MS methods. For proteomics, a reliable, highly multiplexed readout system is required.

  Every biological state can be defined by a complex arrangement of the proteins that make up the “proteome” at a given point in its lifetime. The complete state of the proteome can be defined by listing all the proteins present (including modified variants), their intracellular location and concentration, but such a task is the ability of current single analytical methods. Over. However, attempts have been made to define the state of cells or tissues by identifying and measuring the relative concentrations of a small subset of proteins. For example, Conrads et al. , Analytical Chemistry, 72: 3349-3354 (2000), describes the use of “Accurate Mass Tags” (AMT) for protein identification throughout the proteome. Conrads et al. Shows that a mass spectrometer with sufficient mass accuracy and resolution can be used to detect specific tryptic digest fragments from proteins. Once identified, AMT can be detected directly in the sample by tryptic digests of the protein and high-precision, high-resolution mass spectrometry.

  The concept of precise mass tags is useful for protein discovery and for generating peptide patterns in traditional biological experiments, but it solves the sensitivity problem at the heart of a variety of truly valuable diagnostic protein evaluations. Absent. A valid assessment consisting of AMT requires a sample containing a minimum of 2000 to 10,000 cells to allow reliable reading. This is because many important cellular proteins exist only at the level of 500-5000 molecules per cell. If there are 500 copies of a clinically relevant protein per cell and a valuable clinical sample of a cancer patient contains only 1000 cells, the total number of proteins is 500,000, the limit of detection by conventional mass spectrometry Not reach. Thus, for studies of the proteome after identification of AMT, Conrads et al. The type of measurement proposed by is not suitable for addressing important clinical problems such as cancer diagnosis.

  Disclosed are compositions of one or more analytes and methods of sensitive detection. In general, the method involves the use of special labeling components called multidimensional signals (MDS).

  Thus, according to a first aspect, the present invention is a multi-dimensional signal group comprising one or more sets of reporter signals, and optionally one or more indicator signals, wherein the set of reporter signals comprises a plurality of reporters. The reporter signals in each set have common characteristics, and the common characteristics can distinguish and / or separate reporter signals in each set from molecules lacking the common characteristics, thereby altering the reporter signal Can distinguish each reporter signal in the modified format in each set from any other modified format reporter signal in the set, of the reporter signal and any one or more indicator signals At least one reporter signal due to the common properties Generating a predetermined pattern in situations that can be distinguished and / or separated from molecules lacking the common property, provides a multi-dimensional signal group, characterized in that. When there is more than one set of reporter signal, the common characteristics of each set of reporter signals differ from the common characteristics of another set of reporter signals, and the reporter signal distinguishes the reporter signal from molecules lacking common characteristics and A predetermined pattern is generated in a situation that can be separated.

  According to another aspect, the present invention provides: (a) a set of reporter molecules, each reporter molecule comprising a reporter signal and a decoding tag, the reporter signal having common properties, the common property The signal can be distinguished and / or separated from molecules lacking common properties, the reporter signal can be altered, and each altered reporter signal can be distinguished from all other altered forms of the reporter signal. A reporter molecule, each different reporter molecule having a different decoding tag and different reporter signal, and (b) one or more indicator molecules, each indicator molecule having an indicator signal and a decoding tag A reporter signal and one or more indicator signals By passing characteristics, it provides a kit comprising an indicator molecule to generate a predetermined pattern in situations that can be distinguished and / or separated from molecules lacking the common property of the reporter signals.

  According to yet another aspect, the present invention provides a kit comprising two or more sets of reporter molecules, wherein each reporter molecule comprises a reporter signal and a decoding tag, the reporter signal in each set. Have common properties, which can distinguish and / or separate reporter signals within a set from molecules lacking common properties, the reporter signals can be changed, and the altered format within each set Each reporter signal is distinguishable from all other modified forms of reporter signals in the set, each different reporter molecule having a different decoding tag and a different reporter signal, The common characteristic is different from the common characteristic of another set of reporter signals, By passing characteristics, in situations that can be distinguished and / or separated from molecules lacking the common property of the reporter signal, reporter signals to generate a predetermined pattern.

  In various embodiments of all aspects of the invention, the reporter signal and indicator signal can comprise a peptide, the reporter signal has the same mass to charge ratio, and the at least one indicator signal has the same mass to charge ratio as the reporter signal. Does not have. In some formats, indicator signals do not have common characteristics. The reporter signal and the one or more indicator signals can generate a predetermined pattern in situations where the common property can distinguish and / or separate the reporter signal from molecules lacking the common property. A common property may be a difference in mass-to-charge ratio, the reporter signal can be altered by changing its mass, and the altered form of the reporter signal will change the mass-to-charge ratio of the altered form of the reporter signal. Can be distinguished through. The mass of the reporter signal can be altered by fragmentation. In various embodiments of all aspects of the invention, altering the reporter signal can also alter its charge. The common property may be the mass to charge ratio, the reporter signal can be altered by changing the charge, and the altered form of the labeled protein is mediated by the difference in mass to charge ratio of the altered form of the reporter signal. Are distinguishable.

  In various embodiments of all aspects of the invention, the set is 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 Thus, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more different reporter signals can be provided. The set can comprise 10 or more different reporter signals. The reporter signal may be peptides, oligonucleotides, carbohydrates, polymers, oligopeptides, or peptide nucleic acids.

  In various embodiments of all aspects of the invention, reporter signals can be associated with or linked to specific binding molecules (eg, each reporter signal can be associated with or linked to various specific binding molecules). ). Reporter signals can be associated with or linked to decoding tags (eg, each reporter signal can be associated with or linked to various decoding tags). The reporter signal can be associated with or linked to a protein or peptide. Peptides can have the same amino acid composition, can have the same amino acid sequence, can have different heavy isotope distributions, can have different amino acid sequences, or can have different positions Can have unstable or broken bonds.

  In various embodiments of all aspects of the invention, in some formats, the indicator signal does not have a common characteristic.

  According to another aspect, the present invention provides multiple sets of labeled proteins, wherein each labeled protein comprises a protein or peptide and a reporter signal or indicator signal added to the protein or peptide, the reporter The signals have common properties, and the common properties can distinguish and / or separate labeled proteins comprising the same protein or peptide from molecules lacking common properties. In some embodiments, the reporter signal can be altered, each reporter signal in the altered form can be distinguished from all other altered forms of the reporter signal, and alteration of the reporter signal alters the labeled protein. Each labeled protein in a modified form is distinguishable from all other modified forms of the labeled protein, and the common properties can distinguish and / or separate the labeled protein from molecules lacking common properties In situations, the reporter signal and one or more indicator signals generate a predetermined pattern. In some embodiments, the reporter signal and / or one or more indicator signals generate a predetermined pattern in situations where the common property can distinguish and / or separate the labeled protein from molecules lacking the common property.

  According to another aspect, the present invention provides multiple sets of labeled proteins, wherein each labeled protein comprises a protein or peptide and a reporter signal or indicator signal added to the protein or peptide, the reporter The signal can be changed and each reporter signal in the modified format can be distinguished from all other modified formats of the reporter signal, and changing the reporter signal will change the labeled protein and each modified format reporter signal The labeled protein is distinguishable from all other modified forms of labeled protein, and the reporter signal and one or more indicator signals generate a predetermined pattern.

  According to another aspect, the present invention provides multiple sets of labeled proteins, wherein each labeled protein comprises a protein or peptide and a reporter signal that is added to the protein or peptide, and two reporter signals are provided. Belongs to one of the above set of reporter signals, the reporter signals within each set have common characteristics, the common characteristics of each set of reporter signals differ from the common characteristics of another set of reporter signals, Can distinguish and / or separate labeled proteins comprising the same protein or peptide from molecules lacking common properties, the reporter signal can be altered, and each type of reporter signal in each set can be altered. All other modified forms of the report -Differentiating from the signal, changing the reporter signal changes the labeled protein, and each modified form of the labeled protein is distinguishable from all other modified forms of the labeled protein. In situations where the can be distinguished and / or separated from molecules lacking common properties, the reporter signal produces a predetermined pattern.

  According to yet another aspect, the present invention provides a plurality of sets of labeled proteins, wherein each labeled protein comprises a protein or peptide and a reporter signal added to the protein or peptide, wherein the labeled protein is 2 Being one of more than one set of labeled proteins, the labeled proteins within each set have common characteristics, and the common characteristics of each set of labeled proteins are different from the common characteristics of another set of labeled proteins. In some embodiments, common properties can distinguish and / or separate labeled proteins comprising the same protein or peptide from molecules lacking common properties, the reporter signal can be altered, and each labeled protein within each set can be altered. Each reporter signal in the formatted format is distinguishable from all other modified forms of the reporter signal in the set of labeled proteins in the set, and changing the reporter signal changes the labeled protein and changed in each set In a situation where each labeled protein of the format is distinguishable from all other modified forms of labeled protein in the set, and the common property allows the labeled protein to be distinguished and / or separated from molecules lacking the common property The reporter signal generates a predetermined pattern. In some embodiments, common properties can distinguish and / or separate labeled proteins from molecules lacking common properties, the reporter signal can be altered, and changing the reporter signal can alter the labeled protein and within each set Each of the modified forms of labeled protein is distinguishable from all other unmodified forms of the labeled protein in the set, and the common properties distinguish and / or separate the labeled proteins from molecules that lack common properties In situations where it can, the reporter signal generates a predetermined pattern.

  According to another aspect, the present invention provides a plurality of sets of labeled proteins, wherein each labeled protein comprises a protein or peptide and a reporter signal added to the protein or peptide, and two labeled proteins. The reporter signal belongs to one of the above set of labeled proteins, the reporter signal can be changed, and each modified reporter signal of each labeled protein in each set is associated with every other change of the labeled protein in the set. The reporter signal change modifies the labeled protein, and each modified protein in each set has a different modified protein in each set. The reporter signal generates a predetermined pattern. To.

  According to yet another aspect, the present invention provides a kit comprising a set of reporter molecules and one or more indicator molecules, wherein each reporter molecule comprises a reporter signal and a binding tag, Have common properties, and the common properties can distinguish and / or separate reporter signals from molecules lacking common properties, the reporter signals can be altered, and each reporter signal in the altered form is Differentiated from the altered form of the reporter signal, each different reporter molecule has a different binding tag and different reporter signal, each indicator molecule has an indicator signal and a binding tag, depending on the common properties, the reporter Signal lacks common characteristics In situations that can be distinguished and / or separated from molecules, reporter signals and one or more indicator signals will generate a predetermined pattern.

  According to another aspect, the present invention provides a labeled protein, the labeled protein comprising a protein or peptide and a reporter signal or indicator signal added to the protein or peptide, wherein the labeled protein has a common property, Can distinguish and / or separate labeled proteins from molecules lacking common properties, reporter signals can be altered, reporter signal alterations can alter labeled proteins, and altered forms of labeled proteins can be altered In a situation where the reporter signal and the one or more indicator signals can be distinguished from a form of labeled protein that was not present, and the common property can distinguish and / or separate the labeled protein from molecules lacking the common property, Generation That.

  According to another aspect, the present invention provides a kit comprising a set of reporter molecules, wherein each reporter molecule comprises a reporter signal and a binding tag, the reporter signal comprising two or more sets of reporter signals. The reporter signals in each set have common characteristics, the common characteristics of each set of reporter signals are different from the common characteristics of another set of reporter signals, Can be distinguished and / or separated from molecules lacking properties, the reporter signal can be altered, and each reporter signal in the modified format within each set can be linked to every other modified format reporter in the set Can be distinguished from signals, and each different reporter molecule ; And a case tag and different reporter signals, a common characteristic in situations that can be distinguished and / or separated from molecules lacking the common property of the reporter signal, reporter signals to generate a predetermined pattern.

  In various embodiments of all aspects of the invention, the common property is the mass to charge ratio, the reporter signal can be altered by changing the mass, and the altered form of the labeled protein is altered in the form of the reporter signal. Can be distinguished through the difference in mass-to-charge ratio. The mass of the reporter signal can be altered by fragmentation.

  In various embodiments of all aspects of the invention, the reporter signal can bind to a protein or peptide. The common property allows the labeled protein to be distinguished and / or separated from molecules lacking common properties. The common property may be one or more affinity tags associated with the reporter signal. One or more affinity tags can be associated with a reporter signal. Each labeled protein can comprise a protein or peptide and a reporter signal or indicator signal added to the protein or peptide, the reporter signal comprises a peptide, the reporter signal has the same mass to charge ratio, and the indicator signal is Does not have the same mass to charge ratio as the reporter signal. The reporter signal peptides can have the same amino acid composition or can have the same amino acid sequence. Each reporter signal peptide can contain different distributions of heavy isotopes, can contain different alternative groups, or can have different amino acid sequences. Each reporter signal peptide can have unstable or cleavable bonds at various positions. One or more affinity tags can be associated with a reporter signal.

  According to another aspect, the present invention provides a mixture comprising a set of reporter signal calibrators, one or more indicator signal calibrators, and a set of target protein fragments, wherein each reporter signal calibrator comprises a set of reporter signal calibrators. Shares common properties with target protein fragments within the target protein fragment, and the common properties distinguish and / or separate target protein fragments (eg each of these) and reporter signal calibrators that have common properties from molecules lacking common properties Target protein fragments and reporter signal calibrators that share common properties can correspond to each other, target protein fragments (eg, each of these) can be modified, The protein fragments (eg, each of these) are distinguishable from other altered forms (eg, all other altered forms) of target protein fragments, and reporter signal calibrators (eg, of these Each) is changeable, and each reporter signal calibrator in the modified format is distinguishable from the modified forms of target protein fragments in which the reporter signal calibrator shares common properties, and the target protein fragments having common properties and In situations where the reporter signal calibrator can be distinguished and / or separated from molecules lacking common properties, the reporter signal calibrator and the at least one indicator signal calibrator can generate a predetermined pattern.

  According to another aspect, the present invention provides multiple sets of target protein fragments, wherein each target protein fragment shares common characteristics with a reporter signal calibrator within a set of reporter signal calibrators, and the common characteristics Allows target protein fragments and reporter signal calibrators with common characteristics to be distinguished and / or separated from molecules lacking common characteristics, target protein fragments and reporter signal calibrators that share common characteristics correspond to each other, and target protein fragments are altered The modified form of the target protein fragment can be distinguished from other modified forms of the target protein fragment, the reporter signal calibrator can be modified, and each reporter signal key of the modified form Librators can distinguish reporter signal calibrators from altered forms of target protein fragments that share common properties, and can distinguish and / or separate target protein fragments and reporter signal calibrators that have common properties from molecules that lack common properties Under certain circumstances, the reporter signal calibrator and one or more indicator signal calibrators can generate a predetermined pattern.

  According to another aspect, the present invention provides multiple sets of reporter signal calibrators and one or more indicator signal calibrators, each reporter signal calibrator sharing common characteristics with target protein fragments within a set of target protein fragments, Common properties allow target protein fragments (eg, each of these) and reporter signal calibrators with common properties to be distinguished and / or separated from molecules lacking common properties, and share common protein target protein fragments and reporters Signal calibrators correspond to each other, target protein fragments (eg, each of these) can be changed, and altered forms of target protein fragments (eg, each of these) have been changed to other The reporter signal calibrator (eg, each of these) can be changed and each reporter signal calibrator in the altered format can be distinguished from the target protein fragment of the formula (eg, all other altered formats) The reporter signal calibrator can distinguish from modified forms of target protein fragments that share common properties, and can distinguish and / or separate target protein fragments and reporter signal calibrators that have common properties from molecules that lack common properties. In some situations, the reporter signal calibrator and the at least one indicator signal calibrator can generate a predetermined pattern.

  According to another aspect, the present invention provides a kit for creating a protein signature, the kit comprising: (a) a set of reporter signal calibrators and one or more indicator signal calibrators, each reporter signal calibrator being a set. Share common properties with the target protein fragments within the target protein fragments, wherein the common properties distinguish target protein fragments having common properties (eg, each of these) and reporter signal calibrators from molecules lacking common properties and / or Target protein fragments and reporter signal calibrators that are separable and share common properties correspond to each other, target protein fragments (eg, each of these) can be altered, and altered forms of target protein fragments ( For example, each of these can be distinguished from other modified forms of target protein fragments (eg, all other modified forms), and each reporter signal calibrator can be modified and modified. Each type of reporter signal calibrator is distinguishable from modified forms of target protein fragments where the reporter signal calibrators share common properties, and target protein fragments and reporter signal calibrators with common properties can be distinguished from molecules lacking common properties. The reporter signal calibrator and the at least one indicator signal calibrator can generate a predetermined pattern in a situation that allows differentiation and / or separability. Comprising a Le calibrator, and one or more reagents for creating protein fragment was treated with (b) a protein sample.

  According to another aspect, the present invention provides multiple sets of target protein fragments and one or more indicator signal calibrators, each target protein fragment sharing common characteristics with the reporter signal calibrators of the set of reporter signal calibrators, Common properties allow each target protein fragment and reporter signal calibrator with common properties to be distinguished and / or separated from molecules that lack common properties, and target protein fragments and reporter signal calibrators that share common properties correspond to each other Each target protein fragment can be modified, each modified target protein fragment can be distinguished from all other modified forms of the target protein fragment, and each reporter signal calibrator can be modified Each reporter signal calibrator in the modified format is distinguishable from the modified forms of target protein fragments that the reporter signal calibrator shares common properties, and the target protein fragments and reporter signal calibrators with the common properties are identified. In situations where it is possible to distinguish and / or separate from molecules lacking common properties, the reporter signal calibrator and the at least one indicator signal calibrator can generate a predetermined pattern.

  According to another aspect, the present invention provides multiple sets of reporter signal calibrators, wherein the reporter signal calibrator belongs to one of two or more sets of reporter signal calibrators, and each reporter signal calibrator in each set is 1 A target protein that shares common characteristics with the target protein fragments in one set of target protein fragments, and the common characteristics of each set of reporter signal calibrators are different from the common characteristics of another set of reporter signal calibrators. Fragments (eg, each of these) and reporter signal calibrators can be distinguished and / or separated from molecules that lack common properties, and target protein fragments and reporter signal carriers that share common properties. Blators correspond to each other, target protein fragments (eg, each of these) can be altered, and altered forms of target protein fragments (eg, each of these) can be altered to other altered forms (eg, Can be distinguished from target protein fragments (in all other modified formats), reporter signal calibrators (eg, each of these) can be modified, and each reporter signal calibrator in the modified format is a reporter signal In situations where calibrators are distinguishable from modified forms of target protein fragments that share common properties, allowing target protein fragments and reporter signal calibrators that have common properties to be distinguished and / or separated from molecules lacking common properties, Reporter sig Le calibrator can generate a predetermined pattern.

  According to another aspect, the present invention provides a kit for creating a protein signature, the kit comprising: (a) two or more sets of reporter signal calibrators, wherein the reporter signal calibrators are two or more sets of reporters. Belonging to one of the signal calibrators, each reporter signal calibrator in each set shares common characteristics with the target protein fragments in one set of target protein fragments, and the common characteristics of each set of reporter signal calibrators are different sets Unlike common reporter signal calibrator characteristics, common characteristics make it possible to distinguish and / or separate target protein fragments (e.g. each of these) and reporter signal calibrators that have common characteristics from molecules lacking common characteristics. Target protein fragments and reporter signal calibrators that share characteristics correspond to each other, target protein fragments (eg, each of these) can be modified, and altered forms of target protein fragments (eg, each of these) Is distinguishable from target protein fragments of other modified forms (eg, all other modified forms), and reporter signal calibrators (eg, each of these) can be modified and modified Each type of reporter signal calibrator is distinguishable from modified forms of target protein fragments that the reporter signal calibrator shares common properties, and molecules that lack common properties of target protein fragments and reporter signal calibrators that have common properties. The reporter signal calibrator is capable of generating a predetermined pattern, and (b) one or more reagents for processing the protein sample to produce protein fragments. Is provided.

  According to another aspect, the present invention provides a mixture comprising two or more sets of reporter signal calibrators and a set of target protein fragments, wherein the reporter signal calibrators are more than one set of reporter signals. Belonging to one of the calibrators, each reporter signal calibrator in each set shares common characteristics with the target protein fragments in the set of target protein fragments, and the common characteristic of each set of reporter signal calibrators is another set of reporter signals Unlike common characteristics of calibrators, common characteristics distinguish and / or separate target protein fragments (eg, each of these) and reporter signal calibrators that have common characteristics from molecules that lack common characteristics. Target protein fragments and reporter signal calibrators that share common properties can correspond to each other, target protein fragments (eg, each of these) can be modified, and altered forms of target protein fragments (eg, these Each of which is distinguishable from other modified forms of target protein fragments (eg, all other modified forms), and reporter signal calibrators (eg, each of these) can be modified, Each reporter signal calibrator in the modified format is distinguishable from the target protein fragments in the modified format that the reporter signal calibrator shares common characteristics, and the target protein fragments and reporter signal calibrators that share the common characteristics In the context of the distinguished and / or separable from the Ku molecules, reporter signal calibrators can generate a predetermined pattern.

  According to yet another aspect, the present invention provides multiple sets of target protein fragments, each target protein fragment sharing common characteristics with a reporter signal calibrator within a set of reporter signal calibrators, and more than one reporter signal calibrator. Belonging to one of the set of reporter signal calibrators, the common characteristics of each set of reporter signal calibrators differ from the common characteristics of another set of reporter signal calibrators, and the common characteristics result in target protein fragments having common characteristics (e.g., , Each of these) and reporter signal calibrators can be distinguished and / or separated from molecules lacking common properties, and target protein fragments and reporter signal calibres that share common properties Correspond to each other, target protein fragments (eg, each of these) can be modified, and altered forms of target protein fragments (eg, each of these) can be in other modified forms (eg, Can be distinguished from target protein fragments (in all other modified formats), reporter signal calibrators (eg, each of these) can be modified, and each reporter signal calibrator in the modified format is a reporter signal Reporters in situations where calibrators can distinguish from modified forms of target protein fragments that share common properties, and can distinguish and / or separate target protein fragments and reporter signal calibrators that have common properties from molecules lacking common properties. Signal carry Regulator can generate a predetermined pattern.

  In various embodiments of all aspects of the invention, the set can include a predetermined amount of each reporter signal calibrator. The amount of the at least two reporter signal calibrators can be different. The relative amount of each reporter signal calibrator can be based on the relative amount of each corresponding target protein fragment expected to be in the protein sample. The amount of each of the reporter signal calibrators may be the same. Target protein fragments and reporter signal calibrators can be altered by fragmentation. Target protein fragments and reporter signal calibrators can be altered by cleavage with photolabile amino acids. Target protein fragments and reporter signal calibrators can be fragmented in collision cells. Target protein fragments can be fragmented with aspartate-proline bonds.

  In various embodiments of all aspects of the invention, target protein fragments can be generated by protease digestion of protein samples. Target protein fragments can be generated by digestion of a protein sample with serine protease. The serine protease may be trypsin. Target protein fragments can be generated by cleavage with photolabile amino acids.

  In various embodiments of all aspects of the invention, the common property may be a mass-to-charge ratio, and the target protein fragment and reporter signal calibrator can be altered by changing its mass, its charge, or its mass and charge. And the modified forms of target protein fragments and reporter signal calibrators are distinguishable through the difference in mass to charge ratios of the modified forms of target protein fragments and reporter signal calibrators.

  In various embodiments of all aspects of the invention, the set of reporter signal calibrators is 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more. 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more different reporter signal calibrators. The set of reporter signal calibrators can comprise ten or more different reporter signal calibrators. The target protein fragments of the set are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more , 70 or more, 80 or more, 90 or more, or 100 or more different target protein fragments.

  In various embodiments of all aspects of the invention, the reporter signal calibrator can comprise a peptide, and the peptide has the same mass to charge ratio as the corresponding target protein fragment. A peptide can have the same amino acid composition as the corresponding target protein fragment. The peptide can have the same amino acid sequence as the corresponding target protein fragment. Each peptide can have a different amino acid sequence than the corresponding target protein fragment. Each peptide can have unstable or cleavable bonds at various positions.

  In various embodiments of all aspects of the invention, the reporter signal calibrator may be peptides, oligonucleotides, carbohydrates, polymers, oligopeptides, or peptide nucleic acids. The at least one target protein fragment can comprise at least one modified amino acid. The modified amino acid may be a phosphorylated amino acid, an acylated amino acid, or a glycosylated amino acid. The at least one target protein fragment may be the same as the target protein fragment comprising the modified amino acid except for the modified amino acid.

  According to another aspect, the invention provides multiple sets of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, and a reporter A signal peptide (or an amino acid segment comprising a reporter signal peptide) has common properties, and the common property distinguishes the reporter signal peptide (or amino acid segment comprising a reporter signal peptide) from molecules lacking common properties and / or Alternatively, the reporter signal peptide can be altered. In some embodiments, each reporter signal peptide in an altered form is distinguishable from another reporter signal peptide in an altered form, and the common property distinguishes and / or separates the reporter signal peptide from molecules lacking common properties. In situations where it is possible, the reporter signal peptide and the one or more indicator signal peptides produce a predetermined pattern. In some embodiments, changing the reporter signal peptide changes the amino acid segment, and each amino acid segment in the altered form is distinguishable from another amino acid segment in the altered form, and the common properties make the reporter signal peptide common In situations where it can be distinguished and / or separated from molecules lacking properties, the reporter signal peptide and one or more indicator signal peptides produce a predetermined pattern.

  According to another aspect, the invention provides multiple sets of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, and a reporter signal (or alternatively Amino acid segments) belong to one of two or more sets of reporter signals (or belong to one of two or more sets of amino acid segments), and reporter signal peptides (or amino acid segments) within each set ) Have common characteristics, and the common characteristics of each set of reporter signals (or amino acid segments) are different from the common characteristics of another set of reporter signals (or amino acid segments), depending on the common characteristics Can distinguish and / or separate amino acid segments) from molecules lacking common properties, and reporter signal peptides can be altered, for example, each reporter signal peptide in a modified form can have another reporter signal peptide in a modified form Or a change in the reporter signal peptide alters the amino acid segment, and each amino acid segment in the altered form is distinguishable from another amino acid segment in the altered form, depending on the common properties, the reporter signal peptide In situations where (or amino acid segments) can be distinguished and / or separated from molecules lacking common properties, reporter signal peptides (or amino acid segments) generate a predetermined pattern.

  According to another aspect, the invention provides multiple sets of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, An amino acid segment comprises an amino acid subsegment, each amino acid subsegment comprises a portion of the protein or peptide and all or a portion of a reporter signal peptide or indicator signal peptide, and an amino acid subsegment (eg, a reporter signal peptide All or part of an amino acid segment) have common characteristics, and the common characteristics comprise an amino acid subsegment (eg, all or part of a reporter signal peptide). Amino acid segments) can be distinguished and / or separated from molecules lacking common properties, and the reporter signal peptide can be altered, for example, each altered reporter signal peptide can be altered to another reporter in the altered form. Differentiating from the signal peptide, or changing the reporter signal peptide changes the amino acid subsegment, and each amino acid subsegment in the altered form is distinguishable from another amino acid subsegment in the altered form, with common characteristics Thus, in situations where the amino acid segment comprising all or part of the reporter signal peptide can be distinguished and / or separated from molecules lacking common properties, the amino acid sub-segment produces a predetermined pattern.

  According to another aspect, the invention provides multiple sets of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, wherein each amino acid segment is an amino acid. Each amino acid subsegment comprises one part of the protein or peptide and all or one part of the reporter signal peptide, wherein the amino acid subsegment is one of two or more sets of amino acid subsegments. The amino acid subsegments in each set have common characteristics, and the common characteristics of each set of amino acid subsegments are different from the common characteristics of another set of amino acid subsegments. In some embodiments, the common property can distinguish and / or separate amino acid subsegments comprising all or part of the reporter signal peptide from molecules lacking the common property, and the reporter signal peptide can be altered and altered Each reporter signal peptide in a modified form is distinguishable from another reporter signal peptide in a modified form, and the common properties distinguish amino acid segments comprising all or part of the reporter signal peptide from molecules lacking common properties And / or in situations where they can be separated, amino acid subsegments produce a predetermined pattern. In some embodiments, common properties can distinguish and / or separate amino acid subsegments from molecules lacking common properties, reporter signal peptides can be altered, and alterations in reporter signal peptides can alter amino acid subsegments. Each amino acid subsegment in the altered form is distinguishable from another amino acid subsegment in the altered form, and the amino acid segment comprising all or part of the reporter signal peptide is distinguished from molecules lacking common properties and / or Or, in situations where they can be separated, amino acid subsegments produce a predetermined pattern.

According to another aspect, the invention provides multiple sets of amino acid segments, each amino acid segment comprising a reporter signal peptide and the protein or peptide, wherein the reporter signal peptide is one of two or more sets of reporter signal peptides. Reporter signal peptides in each set have common characteristics, and the common characteristics in each set of reporter signal peptides are different from the common characteristics of another set of reporter signal peptides. Can be distinguished and / or separated from molecules lacking common properties, reporter signal peptides can be changed, and each reporter signal peptide in the altered form can be distinguished from another reporter signal peptide in the altered form There, by the common characteristics, in situations that can be distinguished and / or separated from molecules lacking the common property of the reporter signal peptide, reporter signal peptide to generate a predetermined pattern.
According to another aspect, the present invention provides multiple sets of amino acid segments, each amino acid segment comprising a reporter signal peptide or indicator signal peptide and the protein or peptide, the reporter signal peptide having common characteristics, Can distinguish and / or separate the reporter signal peptide from molecules lacking common properties, the reporter signal peptide can be altered, and each altered reporter signal peptide can have a different reporter signal in the altered form. In situations where the reporter signal can be distinguished from the peptide and the common property can distinguish and / or separate the reporter signal from the molecule lacking the common property, the reporter signal peptide and one or more indicator symbols Null peptide to generate a predetermined pattern.

  According to another aspect, the invention provides cells and multiple sets of cells, each cell or each cell in the set comprising a nucleic acid molecule, each nucleic acid molecule comprising a reporter signal indicator or indicator signal peptide and the protein or peptide The reporter signal peptide has a common property, and the common property can distinguish or separate the reporter signal from molecules lacking the common property, the reporter signal peptide is Each reporter signal peptide in the modified format can be modified from another reporter signal peptide in the modified format, and the common property can distinguish or separate the reporter signal from molecules lacking common properties. Can In the context, the reporter signal peptide and one or more indicator signal peptides will generate a predetermined pattern.

  According to another aspect, the present invention provides a cell comprising a set of nucleic acid molecules, wherein each nucleic acid molecule encodes a reporter signal peptide and an amino acid segment comprising the protein or peptide. The reporter signal peptide belongs to one of two or more sets of reporter signal peptides, the reporter signal peptides in each set have common characteristics, and the common characteristics of each set of reporter signal peptides are different. Unlike the common characteristics of a set of reporter signal peptides, the common characteristics allow the reporter signal peptide to be distinguished and / or separated from molecules lacking the common characteristics, the reporter signal peptide being variable and Each reporter sig In a situation where the reporter signal peptide can be distinguished from another reporter signal peptide in a modified form and the common property can distinguish and / or separate the reporter signal peptide from a molecule lacking the common property, the reporter signal peptide has a predetermined pattern. Is generated.

  According to another aspect, the invention provides multiple sets of cells or organisms, each cell or organism comprising a nucleic acid molecule, each nucleic acid molecule comprising a reporter signal peptide and an amino acid segment comprising the protein or peptide. The reporter signal peptide belongs to one of two or more sets of reporter signal peptides, the reporter signal peptides in each set have common properties, and each set of reporter signal peptides Is different from the common characteristics of another set of reporter signal peptides, which allows the reporter signal peptide to be distinguished and / or separated from molecules lacking the common characteristics, and the reporter signal peptide can be altered, Changed format A reporter signal peptide is distinguishable from another reporter signal peptide in a modified form, and in a situation where the common property can distinguish and / or separate the reporter signal peptide from a molecule lacking the common property, the reporter signal peptide is A predetermined pattern is generated.

  According to another aspect, the present invention provides a living body or a plurality of sets of living bodies, each living body or each living body comprising a nucleic acid molecule, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and the protein or Comprising a nucleotide segment that encodes an amino acid segment comprising a peptide, the reporter signal peptide has common properties, and the common property can distinguish and / or separate the reporter signal peptide from molecules lacking the common property; The reporter signal peptide can be altered, each reporter signal peptide in the altered form can be distinguished from another reporter signal peptide in the altered form, and the common property distinguishes the reporter signal peptide from molecules lacking the common property. And / or in situations which may be separated, the reporter signal peptide and one or more indicator signal peptides will generate a predetermined pattern.

  In various embodiments of all aspects of the invention, each nucleic acid molecule can further comprise an expression sequence, and the expression sequence can be operably linked to the nucleotide segment such that the amino acid segment is expressed. The expression sequence of each nucleic acid molecule may be different. Various expression sequences may be differentially adjusted. The expression sequence may be similarly adjusted. The plurality of expression sequences may be an expression sequence of a gene expressed as part of the same expression cascade, or may be obtained from the gene. The expression sequence can comprise a translational expression sequence and / or a transcriptional expression sequence. The amino acid segment can be expressed in vitro or in vivo. Amino acid segments can be expressed in cell culture. The expression sequence of each nucleic acid molecule may be the same. The expression sequences of at least two nucleic acid molecules may be different or the same. Each nucleic acid molecule can further comprise a replication sequence, which allows the nucleic acid molecule to replicate.

  In various embodiments of all aspects of the invention, the nucleic acid molecule can be replicated in vitro or in vivo. Nucleic acid molecules can replicate in cell culture. Each nucleic acid molecule can further comprise an integrated sequence, which can integrate the nucleic acid molecule into other nucleic acids. Nucleic acid molecules can be integrated into a chromosome (eg, at a predetermined location). Nucleic acid molecules can be made by replicating nucleic acids in one or more nucleic acid samples. Nucleic acids can be replicated using a pair of primers, and each first primer of the primer pair used to create the nucleic acid molecule can comprise a nucleotide sequence that encodes a reporter signal peptide. . Each first primer can further comprise an expression sequence. The nucleotide sequence of each first primer also encodes an epitope tag.

  In various embodiments of all aspects of the invention, each amino acid segment can further comprise an epitope tag. The epitope tag of each amino acid segment may be different or the same. The epitope tags of at least two amino acid segments may be different or the same. The reporter signal peptide of each amino acid segment may be different or the same. The reporter signal peptides of at least two amino acid segments may be different or the same.

  In various embodiments of all aspects of the invention, the nucleic acid molecule may be in a cell or cell line. Each nucleic acid molecule may be in a different cell (or cell line) or in the same cell (or cell line). The nucleic acid molecule may be in a living body. Each nucleic acid molecule may be in a different living body or in the same living body. Nucleic acid molecules can be integrated into the chromosome of a cell or organism (eg, at a predetermined location). The chromosome may be an artificial chromosome. The nucleic acid molecule may be a plasmid or may be integrated into a plasmid. The nucleic acid molecule may be in a cell of a living body (for example, almost all of a living body cell or a part of a living body cell). Amino acid segments can be expressed in almost all cells of a living organism or in a portion of cells of a living organism.

  In various embodiments of all aspects of the invention, the protein or peptide of each amino acid segment may be different or the same. The protein or peptide of the at least two amino acid segments may be different or the same. The protein or peptide may be related, may be a protein produced in the same cascade, may be a protein in the same enzymatic pathway, or is a protein expressed in the same context. It may be a protein associated with the same disease or a protein associated with the same cell type or the same tissue type.

  In various embodiments of all aspects of the invention, the nucleotide segment may encode a plurality of amino acid segments each comprising a reporter signal peptide or indicator signal peptide and the protein or peptide. The protein or peptide of the at least two amino acid segments of one of the nucleotide segments may be different. The protein or peptide of the amino acid segment of one of the nucleotide segments may be different. The protein or peptide of at least two amino acid segments of each nucleotide segment may be different. The protein or peptide of the amino acid segment of each nucleotide segment may be different.

  In various embodiments of all aspects of the invention, the set of nucleic acid molecules can consist of a single nucleic acid molecule. A nucleic acid molecule can comprise a plurality of nucleotide segments, each encoding an amino acid segment. The amino acid segment can comprise a cleavage site near the junction between the reporter signal peptide and the protein or peptide. The cleavage site may be a trypsin cleavage site. The cleavage site may be at the junction between the reporter signal peptide and the protein or peptide. Each amino acid segment can further comprise a self-cleaving segment. The self-cleaving segment may be between the reporter signal peptide and the protein or peptide. The self-cleaving segment may be an intein segment.

  In various embodiments of all aspects of the invention, the amino acid segment may be a protein or peptide. The set of amino acid segments can be composed of a single amino acid segment, which comprises a plurality of reporter signal peptides.

  In various embodiments of all aspects of the invention, each cell or organism can further comprise additional nucleic acid molecules. The set of cells can consist of a single cell, and the cell comprises a plurality of nucleic acid molecules. A set can consist of a single cell, a cell comprising a set of nucleic acid molecules, a set of nucleic acid molecules consisting of a single nucleic acid molecule, and a nucleic acid molecule encoding a plurality of nucleic acid segments. Similarly, a set of organisms can consist of a single organism, and the organism comprises a plurality of nucleic acid molecules. A set can consist of a single organism, the organism comprises a set of nucleic acid molecules, the set of nucleic acid molecules consists of a single nucleic acid molecule, and the nucleic acid molecule encodes a plurality of nucleic acid segments.

  According to another aspect, the present invention provides: (a) in each sample of one sample or a plurality of samples, the reporter signal in each set has a common characteristic, and the common characteristic of the reporter signal in each set Separating two or more sets of reporter signals that differ from the common characteristics of the set of reporter signals from molecules lacking the common characteristics; and (b) identifying a predetermined pattern generated by the reporter signal and the one or more indicator signals. Providing a method comprising: (c) altering a reporter signal that produces a predetermined pattern; and (d) detecting and distinguishing the altered form of the reporter signal from each other.

  According to another aspect, the present invention provides (a) having one or more reporter signals and one or more indicator signals each having a common characteristic in one sample or each of a plurality of samples. Separating from the missing molecule; (b) identifying a predetermined pattern generated by the reporter signal; (c) modifying the reporter signal that generates the predetermined pattern; and (d) a modified form of the reporter. Detecting the signals and distinguishing them from each other.

  In some forms of the invention, indicator signals do not have common characteristics. In situations where the common property allows the reporter signal to be distinguished and / or separated from molecules lacking the common property, the set of reporter signals and one or more indicator signals can generate a predetermined pattern. A common property may be a mass to charge ratio, where the reporter signal can be altered by changing its mass, and the altered form of the reporter signal is mediated by the difference in mass to charge ratio of the altered form of the reporter signal. Can be distinguished. The mass of the reporter signal can be altered by fragmentation. The set of reporter signals is 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, There can be 70 or more, 80 or more, 90 or more, or 100 or more different reporter signals. The set of reporter signals can comprise 10 or more different reporter signals.

  In various embodiments of all aspects of the invention, the reporter signal may be peptides, oligonucleotides, carbohydrates, polymers, oligopeptides, or peptide nucleic acids. Reporter signals can be associated with or bound to specific binding molecules, and each reporter signal can be associated with or bound to various specific binding molecules. Reporter signals can be associated with or associated with decoding tags, and each reporter signal can be associated with or associated with various decoding tags.

  In various embodiments of all aspects of the invention, the method can further comprise the step of associating a reporter signal with one or more analytes prior to step (a), wherein each reporter signal is Can be associated with or bound to various specific binding molecules, each specific binding molecule can specifically interact with a different one of the analytes, and the reporter signal can interact with the specific binding molecule and the analyte Can be associated with the specimen via Steps (a)-(d) can be repeated one or more times, each time with a different set of one or more reporter signals (the same or different set of indicator signals can be used each time If possible). Prior to step (a), different sets of reporter signals can be associated with different samples.

  In various embodiments of all aspects of the invention, the different sets of reporter signals can each comprise the same reporter signal. Multiple sets of reporter signals can each comprise a single reporter signal. Not all reporter signals in the set need to be distinguished and / or separated from molecules lacking common properties, not all reporter signals need to be changed, and all modified forms of reporter signals can be detected simultaneously. There is no need to All reporter signals in a set can be distinguished and / or separated from molecules lacking common properties, all reporter signals can be modified, and all modified forms of reporter signals can be detected at various times .

  In various embodiments of all aspects of the invention, steps (a)-(d) can be performed separately for each reporter signal. The reporter signal can comprise a peptide, and the peptides have the same mass to charge ratio. The peptides can have the same amino acid composition or the same amino acid sequence. Each peptide can contain different distributions of heavy isotopes, have different amino acid sequences, or have unstable or cleavable bonds at different positions.

  In various embodiments of all aspects of the invention, the set of reporter signals and one or more indicator signals are predetermined in situations where the common property allows the reporter signal to be distinguished and / or separated from molecules lacking the common property. A pattern can be generated. Not all reporter signals need to be distinguished and / or separated from molecules lacking common properties, all reporter signals need not be changed, and all modified forms of reporter signals need to be detected simultaneously Absent. All reporter signals can be distinguished and / or separated from molecules lacking common properties, all reporter signals can be altered, and all altered forms of reporter signals can be detected at various times.

  According to another aspect, the present invention provides (a) separating one or more labeled proteins, each labeled protein comprising a protein or peptide and a reporter signal added to the protein or peptide; The signal belongs to one of two or more sets of reporter signals, each reporter signal has a common characteristic, and the common characteristic of each set of reporter signals is different from the common characteristic of another set of reporter signals (e.g. A common property (within each of the one or more samples) can distinguish and / or separate labeled proteins comprising the same protein or peptide from molecules lacking the common property; (a) one or more Separating labeled proteins and each labeled protein is a protein Or a reporter signal or indicator signal added to a protein or peptide, each reporter signal having a common property, eg, within each of one or more samples, by a common property, A step of distinguishing and / or separating a labeled protein comprising the same protein or peptide from a molecule lacking common properties, or (a) separating one or more labeled proteins from another molecule, wherein the labeled protein Can be obtained from one or more samples, each labeled protein comprising a protein or peptide and a reporter signal or indicator signal added to the protein or peptide; (b) a reporter signal; and If present, identifying a predetermined pattern generated by the one or more indicator signals; (c) changing the labeled protein by changing the reporter signal that generates the predetermined pattern; and (d) being changed. Detecting different types of labeled proteins and distinguishing them from each other.

  According to another aspect, the invention comprises (a) separating a set of labeled proteins, each labeled protein comprising a protein or peptide and a reporter signal or indicator signal added to the protein or peptide; Each labeled protein has a common property, wherein the common property allows a labeled protein comprising the same protein or peptide to be distinguished and / or separated from molecules lacking the common property; and (b) a reporter signal and one or more Identifying a predetermined pattern generated by the indicator signal; (c) changing the labeled protein by changing a reporter signal that generates the predetermined pattern; and (d) detecting the changed form of the labeled protein. Distinguish between each other To provide a method of and a-up.

  According to another aspect, the invention comprises (a) modifying one or more labeled proteins, wherein the labeled protein comprises a protein or peptide and a reporter signal or indicator signal added to the protein or peptide; The labeling protein can be altered by changing the reporter signal, and (b) the altered form of the labeled protein from the unmodified form of the tagged protein or when two or more of the labeled proteins are altered Detecting and distinguishing from each other, wherein the reporter signal and / or the one or more indicator signals generate a predetermined pattern. In some embodiments, the method is used to detect a protein or peptide.

  According to another aspect, the present invention provides (a) separating a set of labeled proteins, each labeled protein comprising a protein or peptide and a reporter signal added to the protein or peptide, wherein the labeled protein Belongs to one of two or more sets of labeled proteins, each labeled protein has a common characteristic, and the common characteristic of each set of labeled proteins is different from the common characteristic of another set of labeled proteins. Distinguishing and / or separating labeled proteins comprising the same protein or peptide from molecules lacking common properties; (b) identifying a predetermined pattern generated by the reporter signal; and (c) a predetermined pattern Labeling by changing the reporter signal generated Providing and changing the click quality, a method of and a distinguishing step with each other to detect the type of the labeled protein has been changed (d).

  According to another aspect, the present invention provides a method for detecting a protein, the method comprising detecting a labeled protein, wherein the labeled protein is a protein or peptide and a reporter signal, or a reporter attached to the protein or peptide. A signal or indicator signal, wherein the labeled protein is altered by altering the reporter signal and detecting the altered form of the labeled protein, wherein the labeled protein is altered by altering the reporter signal And identifying the protein based on the characteristics of the labeled protein and the modified form of the labeled protein, wherein the reporter signal, and one or more indicator signals, if any, And a step of generating a.

  According to another aspect, the invention comprises (a) separating one or more labeled proteins from another molecule, wherein the labeled proteins can be obtained from one or more samples, each labeled protein being a protein Or comprising a peptide and a reporter signal or indicator signal added to the protein or peptide; (b) identifying a predetermined pattern generated by the reporter signal and / or one or more indicator signals; One or more samples detected by altering a labeled protein by altering a reporter signal that produces a predetermined pattern; and (d) detecting and distinguishing the altered form of the labeled protein from each other. In protein and peptide Providing Bei proteins and peptides catalog.

  In various embodiments of all aspects of the invention, the common property may be mass to charge ratio, the reporter signal can be altered by changing its mass, and the altered form of labeled protein has been altered. A distinction can be made through differences in the mass-to-charge ratio of the labeled proteins of the form. The mass of the reporter signal can be altered by fragmentation. Changing the reporter signal also changes its charge. The common property may be the mass-to-charge ratio, the reporter signal can be altered by changing the charge, and the altered form of labeled protein can be distinguished via the altered form of the reporter signal's mass-to-charge ratio It is. The set of labeled proteins is 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 More than 80, 90 or more, or 100 or more different reporter signals can be provided. The set of labeled proteins can comprise 10 or more different reporter signals.

  In various embodiments of all aspects of the invention, the reporter signal may be peptides, oligonucleotides, carbohydrates, polymers, oligopeptides, or peptide nucleic acids. The reporter signal can be bound to a protein or peptide. Steps (a)-(d) can be performed separately for each labeled protein. The method further comprises adding a reporter signal to one or more proteins, one or more peptides, or one or more proteins and peptides prior to step (a). The steps can be repeated one or more times, each time with a different set of one or more reporter signals (the same or different set of indicator signals can be used each time). Prior to step (a), different sets of reporter signals can be added to proteins or peptides in different samples. Each different set of reporter signals can comprise the same reporter signal. Multiple sets of reporter signals can each comprise a single reporter signal.

  In various embodiments of all aspects of the invention, not all of the labeled proteins in the set need to be distinguished and / or separated from molecules lacking common properties, not all reporter signals need to be changed, all It will be appreciated that the modified forms of labeled proteins need not be detected simultaneously. All labeled proteins in the set can be distinguished and / or separated from molecules lacking common properties, all reporter signals can be changed, and all modified forms of labeled protein are detected at various times be able to. Steps (a)-(d) can be performed separately for each reporter signal.

  In various embodiments of all aspects of the invention, the common property may be one or more affinity tags associated with the reporter signal. One or more affinity tags can be associated with a reporter signal. The collection of detected and modified forms of labeled protein can constitute a catalog of proteins. Steps (a)-(d) can be performed separately for each sample. Different samples can be obtained from the same protein sample. Different samples can be obtained at different time points, can be obtained from the same type of organism, can be obtained from the same type of tissue, can be obtained from the same organism, or can be obtained at different time points .

  In various embodiments of all aspects of the invention, the various samples may be samples from different organisms, different types of tissues, different species of organisms, different species of organisms, or different cell compartments. The method further comprises identifying or creating a protein or peptide that corresponds to a protein or peptide that is in one sample but not in another sample. The method further comprises determining the relative amount of protein or peptide in the various samples.

  In various embodiments of all aspects of the invention, the presence, amount, presence and amount, or absence pattern of one labeled protein in a sample constitutes a catalog of proteins in the sample. The presence, amount, presence and amount, or absence pattern of labeled protein in the second sample constitutes a catalog of proteins in the second sample, and the catalog of proteins in the first sample is the first catalog. And the catalog of proteins in the second sample is a second catalog, and the method further comprises comparing the first catalog with the second catalog.

  In various embodiments of all aspects of the invention, each labeled protein can comprise a protein or peptide and a reporter signal or indicator signal added to the protein or peptide, the reporter signal comprising a peptide, The signal has the same mass to charge ratio and the indicator signal does not have the same mass to charge ratio as the reporter signal. The reporter signal peptide can have the same amino acid composition or the same amino acid sequence. Each reporter signal peptide can contain different distributions of heavy isotopes, can contain different distributions of alternative groups, can have different amino acid sequences, or is unstable or cleaved at different positions. Can have a bond.

  In various embodiments of all aspects of the invention, the method further comprises detecting an unmodified form of the labeled protein. Labeled protein and modified form of labeled protein can be detected by detecting the mass to charge ratio of the labeled protein and the mass to charge ratio of the modified form of the labeled protein or the mass to charge ratio of the modified form of the reporter signal It is. The method further includes, prior to step (a), one or more reporter signals and one or more indicator signals and one or more proteins, one or more peptides from one or more samples, or one The method further comprises associating the above proteins and peptides with the reporter signal and the one or more indicator signals generating a predetermined pattern.

  In various embodiments of all aspects of the invention, the different sets of reporter signals can each comprise the same reporter signal. Each reporter signal or each labeled protein can have common properties, which can distinguish and / or separate labeled proteins with the same protein or peptide from molecules lacking common properties. One or more labeled proteins can be obtained from a single sample. A single labeled protein can be distinguished and / or separated from another molecule. Multiple labeled proteins can be distinguished and / or separated from other molecules.

  In various embodiments of all aspects of the invention, the detected altered form of the labeled protein constitutes a catalog of proteins in the sample. One or more labeled proteins can be obtained from each of the plurality of samples. A single labeled protein obtained from each sample can be distinguished and / or separated from another molecule. The plurality of labeled proteins obtained from each sample can be distinguished and / or separated from other molecules. The detected and modified forms of labeled protein obtained from each sample can constitute a catalog of proteins within the sample.

  According to another aspect, the present invention provides a method for creating a protein signature, the method comprising: (a) processing a protein sample to produce a protein fragment, wherein the protein fragment is a set of target protein fragments. A target protein fragment (eg, each of these) can be altered, and the altered form of the target protein fragment is distinguishable from other altered forms of the target protein fragment; (b) ) Mixing the target protein fragment with a set of reporter signal calibrators and one or more indicator signal calibrators, each target protein fragment sharing common characteristics with at least one reporter signal calibrator, Target tamper with common characteristics Fragments (e.g., each of these and the reporter signal calibrator can be distinguished and / or separated from molecules lacking common properties, target protein fragments and reporter signal calibrators that share common properties correspond to each other, and reporter The signal calibrator (eg, each of these) can be modified, and each modified reporter signal calibrator can be distinguished from the modified forms of target protein fragments that the reporter signal calibrator shares common characteristics with. (C) separating the target protein fragment and reporter signal calibrator from another molecule based on the common properties of the target protein fragment and reporter signal calibrator; and (d) repo Identifying a predetermined pattern generated by the data signal calibrator and the one or more indicator signal calibrators; (e) changing the target protein fragment and reporter signal calibrator that generated the predetermined pattern; and (f) changing Detecting a target protein fragment of a modified format and a reporter signal calibrator, wherein the presence, absence, amount, or presence and amount of the target protein fragment of a modified format is obtained from the target protein fragment of a modified format. Indicates the presence, absence, amount, or presence and amount of the protein sample of the target protein fragment, and the presence, absence, amount, or presence and amount of the target protein fragment in the protein sample indicates the protein signature of the protein sample Comprising the step of configuring, the.

  According to another aspect, the present invention provides a method of generating a protein signature, the method comprising: (a) processing a protein sample to generate a protein fragment, wherein the protein fragment is a set of target proteins. Comprising a fragment, wherein the target protein fragment is alterable, wherein the altered form of the target protein fragment (eg, each of these) is distinguishable from other altered forms of the target protein fragment; b) mixing the target protein fragment with two or more sets of reporter signal calibrators, wherein the reporter signal calibrator belongs to one of the two or more sets of reporter signal calibrators, and each target protein fragment is at least With one reporter signal calibrator Shared characteristics, the common characteristics of each set of reporter signal calibrators are different from the common characteristics of another set of reporter signal calibrators, and depending on the common characteristics, target protein fragments (for example, each of them) and reporter signals that have a common characteristic Calibrators can be distinguished and / or separated from molecules that lack common properties, target protein fragments and reporter signal calibrators that share common properties correspond to each other, and reporter signal calibrators (for example, each of these) can be changed Each reporter signal calibrator in the modified format is distinguishable from the modified form of the target protein fragment where the reporter signal calibrator shares common properties; Separating the target protein fragment and the reporter signal calibrator from another molecule based on the common properties of the target protein fragment and the reporter signal calibrator; and (d) identifying a predetermined pattern generated by the reporter signal calibrator; A step of changing a target protein fragment and a reporter signal calibrator that have generated a predetermined pattern; and a step of detecting a changed type of target protein fragment and a reporter signal calibrator, the target protein fragment having the changed format. Presence, absence, amount, or presence and amount indicates the presence, absence, amount, or presence and amount of a protein sample of the target protein fragment that results in a modified form of the target protein fragment. The presence, absence, amount, or presence and amount of the target protein fragment in the protein sample comprises the protein signature of the protein sample.

  According to another aspect, the invention provides a method for creating a protein signature, the method comprising identifying a reporter signal and a predetermined pattern generated by a calibrator and one or more indicator signal calibrators, and modified Detecting a target protein fragment of a format and a reporter signal calibrator, wherein the modified format of the target protein fragment (eg, each of these) is changed to another modified format (eg, all other modified Each target protein fragment shares a common characteristic with at least one reporter signal calibrator, and the common characteristic causes a common target protein fragment (e.g., each of these) and Porter signal calibrators can be distinguished and / or separated from molecules lacking common properties, target protein fragments and reporter signal calibrators that share common properties correspond to each other, and each modified form of reporter signal calibrator is The presence, absence, amount, or presence and amount of the altered form of the target protein fragment is distinguishable from the altered form of the target protein fragment where the signal calibrator shares common properties. Indicates the presence, absence, amount, or presence and amount of the protein sample of the target protein fragment from which the fragment is obtained, and the presence, absence, amount, or presence and amount of the target protein fragment in the protein sample constitutes the protein signature of the protein sample. The method comprising the steps of, comprising a. In some embodiments, the reporter signal calibrator and the one or more indicator signal calibrators generate a predetermined pattern in situations where the common characteristic can distinguish and / or separate the reporter signal calibrator from molecules lacking the common characteristic.

  According to another aspect, the present invention provides a method of creating a protein signature, the method comprising: identifying a predetermined pattern generated by a reporter signal calibrator; and modified forms of target protein fragments and reporter signal calibrators. Wherein the modified form of the target protein fragment (eg, each of these) is the target of another modified form (eg, all other modified forms). The reporter signal calibrator belongs to one of two or more sets of reporter signal calibrators, each target protein fragment shares common characteristics with at least one reporter signal calibrator, Reporter The common characteristics of the null calibrators are different from the common characteristics of another set of reporter signal calibrators, and the common characteristics result in target protein fragments that have common characteristics (for example, each of these and the reporter signal calibrator from molecules that lack common characteristics). Target protein fragments and reporter signal calibrators that can be distinguished and / or separated and share common properties correspond to each other, and each type of reporter signal calibrator in the modified form is modified so that the reporter signal calibrator shares the common properties. The presence, absence, amount, or presence and amount of a modified form of the target protein fragment is distinguishable from the different forms of the target protein fragment, and the target protein from which the modified form of the target protein fragment is obtained Indicate the presence, absence, amount, or presence and amount of a piece of protein sample, and the presence, absence, amount, or presence and amount of a target protein fragment within the protein sample constitutes the protein signature of the protein sample. The reporter signal calibrator generates a predetermined pattern in situations where the common property can distinguish and / or separate the reporter signal calibrator from molecules lacking the common property.

  According to another aspect, the present invention provides a method of generating a protein signature, the method comprising: (a) processing a protein sample to generate a protein fragment, wherein the protein fragment is a set of target proteins. The target protein fragments (eg, each of these) can be modified, and the modified forms of the target protein fragments (eg, each of these) can be in other modified forms (eg, all Distinguishable from target protein fragments of other modified forms), (b) separating the target protein fragment from another protein fragment in the protein sample, (c) the target protein fragment and optionally Identifies the predetermined pattern generated by one or more indicator signal calibrators. (D) changing the target protein fragment that generated the predetermined pattern; and (e) detecting the altered form of the target protein fragment, wherein the altered form of the target protein fragment Presence, absence, amount, or presence and amount indicates the presence, absence, amount, or presence and amount of a protein sample of the target protein fragment that results in a modified form of the target protein fragment, and the target protein fragment in the protein sample The presence, absence, amount, or presence and amount comprises a protein signature of the protein sample.

  According to another aspect, the present invention provides a method of creating a protein signature, the method comprising: (a) separating a plurality of target protein fragments from other protein fragments in a protein sample; and (b) a target Identifying a protein fragment and optionally a predetermined pattern generated by one or more indicator signal calibrators; (c) modifying the target protein fragment that generated the predetermined pattern; (d) a modified form of the target Detecting the protein fragment, wherein the presence, absence, amount, or presence and amount of the altered form of the target protein fragment is present in the presence of a protein sample of the target protein fragment from which the altered form of the target protein fragment is obtained Indicates the absence, quantity, or presence and quantity within the protein sample The presence of protein fragments comprises absent, a step amount or presence and amount, are constituting a protein signatures of a protein sample.

  According to another aspect, the present invention provides a method for analyzing a protein sample, the method comprising: (a) mixing a protein sample with a predetermined amount of a reporter signal calibrator and one or more indicator signal calibrators. The protein sample has a known amount of protein, the protein sample comprises a target protein fragment, the target protein fragment can be altered, the reporter signal calibrator can be altered, and the altered form of the reporter signal calibrator is (C) identifying a predetermined pattern generated by the reporter signal calibrator and one or more indicator signal calibrators; Comprising the step of changing the target protein fragments and reporter signal calibrators to generate a pattern, and detecting the target protein fragments and reporter signal calibrators of the type has changed (d).

  According to another aspect, the present invention provides a method for analyzing a protein sample, the method comprising: (a) mixing a protein sample with a predetermined amount of two or more reporter signal calibrators, the method comprising: Has a known amount of protein, the protein sample has a target protein fragment, the target protein fragment can be modified, the reporter signal calibrator can be modified, and the modified form of the reporter signal calibrator can be modified (B) identifying a predetermined pattern generated by the reporter signal calibrator; (c) changing the target protein fragment and reporter signal calibrator that generated the predetermined pattern. When, and a step of detecting a target protein fragments and reporter signal calibrators of the type has changed (d).

  According to another aspect, the present invention provides a method for analyzing a protein sample, the method comprising: (a) processing the protein sample to produce protein fragments, wherein the protein sample comprises a known amount of protein. A protein sample comprises a target protein, and the protein fragment comprises a target protein fragment obtained from the target protein; (b) a protein sample, a predetermined amount of reporter signal calibrator and one or more indicator signal calibrators; The target protein fragment can be altered, the reporter signal calibrator can be altered, and the altered form of the reporter signal calibrator can be distinguished from the altered form of the target protein fragment. (C) identifying a predetermined pattern generated by the reporter signal calibrator and the one or more indicator signal calibrators; (d) changing the target protein fragment and reporter signal calibrator that generated the predetermined pattern; e) detecting the altered form of the target protein fragment and the reporter signal calibrator.

  According to another aspect, the present invention provides a method for analyzing a protein sample, the method comprising: (a) processing the protein sample to produce protein fragments, wherein the protein sample comprises a known amount of protein. The protein sample comprises a target protein, the protein fragment comprises a target protein fragment obtained from the target protein, and (b) a step of mixing the protein sample with a predetermined amount of two or more reporter signal calibrators The reporter signal calibrator belongs to one of the two or more sets of reporter signal calibrators, the target protein fragment can be altered, the reporter signal calibrator can be altered, and the reporter signal in a modified form Calibre The data is distinguishable from the altered form of the target protein fragment, (c) identifying the predetermined pattern generated by the reporter signal calibrator, and (d) the target protein fragment and reporter signal that generated the predetermined pattern. Modifying the calibrator; and (e) detecting the altered form of the target protein fragment and the reporter signal calibrator.

  In some formats, indicator signal calibrators do not have common characteristics. In situations where the common property allows the reporter signal calibrator to be distinguishable and / or separable from molecules lacking the common property, the reporter signal calibrator and one or more indicator signal calibrators can generate a predetermined pattern. Steps (e) and (f) can be performed simultaneously. Modified forms of target protein fragments can be detected using mass spectrometry. Steps (c), (d), (e), and (f) can be performed using a tandem mass spectrometer.

  In various embodiments of all aspects of the invention, the tandem mass spectrometer can comprise a first stage and a last stage, and step (c) is narrow using the first stage of the tandem mass spectrometer. Ions can be selected within the mass charge range, step (e) can be performed by collision with a gas, and step (f) can be performed using the last stage of the tandem mass spectrometer. . The first stage of the tandem mass spectrometer may be a quadrupole mass filter. The final stage of the tandem mass spectrometer may be a time of flight analyzer. The tandem mass spectrometer may be the last stage time-of-flight analyzer. The mass charge range can be altered to cover the respective mass charge range of the target protein fragment.

  In various embodiments of all aspects of the invention, a predetermined amount of each reporter signal calibrator can be mixed with the target protein fragment, and the amount of each altered form of the reporter signal calibrator detected can be altered. It is understood that a criterion for assessing the amount of the corresponding target protein fragment can be provided. The amount of the at least two reporter signal calibrators can be different. The relative amount of each reporter signal calibrator can be based on the relative amount of each corresponding target protein fragment expected in the protein sample. The amount of each reporter signal calibrator can be the same.

  In various embodiments of all aspects of the invention, the target protein fragment and the reporter signal calibrator can be altered by fragmentation or cleavage with a photolabile amino acid. Target protein fragments and reporter signal calibrators can be fragmented in collision cells or asparagine-proline bonds. Protein fragments can be generated by protease digestion of protein samples. The protease may be a serine protease (eg, trypsin). Protein fragments can be made by digestion of protein samples with factor Xa or enterokinase or by cleavage of photolabile amino acids.

  In various embodiments of all aspects of the invention, the common property may be a mass to charge ratio, and the target protein fragment and reporter signal calibrator can be altered by changing its mass, its charge, or its mass and charge. The modified forms of target protein fragments and reporter signal calibrators are distinguishable through the difference in mass-to-charge ratio of the modified forms of target protein fragments and reporter signal calibrators. The target protein fragments of the set are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more , 70 or more, 80 or more, 90 or more, or 100 or more different target protein fragments. The set of target protein fragments can comprise 10 or more different target protein fragments.

  In various embodiments of all aspects of the invention, the set of reporter signal calibrators is 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more different reporter signal calibrators. The reporter signal calibrator can comprise a peptide, which has the same mass to charge ratio as the corresponding target protein fragment.

  In various embodiments of all aspects of the invention, the peptide can have the same amino acid composition as the corresponding target protein fragment. The peptide can have the same amino acid sequence as the corresponding target protein fragment. Each peptide can have a different amino acid sequence than the corresponding target protein fragment. Each peptide can have unstable or cleavable bonds at various positions. Reporter signal calibrators may be peptides, oligonucleotides, carbohydrates, polymers, oligopeptides, or peptide nucleic acids.

  In various embodiments of all aspects of the invention, the method further comprises comparing the protein signature with one or more other protein signatures. The at least one target protein fragment can comprise at least one modified amino acid. The modified amino acid may be a phosphorylated amino acid, an acylated amino acid, or a glycosylated amino acid. The at least one target protein fragment may be the same as the target protein fragment comprising the modified amino acid except for the modified amino acid.

  In various embodiments of all aspects of the invention, the method further comprises performing steps (a)-(f) on a plurality of protein samples. The method further comprises identifying a difference between protein signatures made from the protein sample. The method further comprises performing steps (a)-(f) on the control protein sample and identifying a difference between protein signatures made from the protein sample and the control protein sample. The difference may be a difference in the presence, amount, presence and amount, or absence of the target protein fragment in the protein sample and the control protein sample.

  In various embodiments of all aspects of the invention, steps (a)-(f) can be performed on a control protein sample and a test protein sample, and the source of the test protein sample or test protein sample is of step (a). Prior to processing, one or more protein molecules in a test protein sample can be processed to be destroyed, discarded, or removed, and the target protein fragment corresponding to the destroyed, discarded, or removed protein molecule is a test protein. Made from a control protein sample rather than a sample. The test protein sample can be processed to destroy, discard, or remove one or more protein molecules in the test protein sample. One or more protein molecules in the test sample can be removed by separating the one or more protein molecules from the test protein sample. One or more protein molecules can be separated by affinity separation. The source of the test protein sample can be processed to destroy, discard, or remove one or more protein molecules in the test protein sample. The processing of the source can be performed by exposing the cells from which the test sample is obtained to a compound, composition, or situation that reduces or eliminates the expression of one or more genes.

  In various embodiments of all aspects of the invention, the method further comprises identifying a difference between target protein fragments in the control protein sample and the test protein sample. The method can further comprise identifying differences between target protein fragments in the protein sample. Multiple protein samples can be generated by a separation procedure, which includes liquid chromatography, gel electrophoresis, two-dimensional chromatography, two-dimensional gel electrophoresis, isoelectric focusing, thin layer chromatography, centrifugation, filtration, ion chromatography, Immonoaffinity chromatography, membrane separation, or a combination thereof. Protein samples may be different fragments or samples made with the same separation procedure.

  In various embodiments of all aspects of the invention, the method further comprises performing steps (a)-(f) on the second protein sample. The second protein sample may be a sample from the same type of organism as the first protein sample. The second protein sample may be a sample from the same type of tissue as the first protein sample. The second protein sample may be a sample from the same organism as the first protein sample. The second protein sample can be obtained at a different time than the first protein sample. The second protein sample may be a sample from a living body different from the first protein sample. The second protein sample may be a sample from a different type of tissue than the first protein sample. The second protein sample may be a sample from a different species of organism than the first protein sample. The second protein sample may be a sample from a different species of organism than the first protein sample. The second protein sample may be a sample from a different cell compartment than the first protein sample.

  In various embodiments of all aspects of the invention, the method further comprises creating a second protein signature from the second protein sample and comparing the first protein signature and the second protein signature. The difference between the first and second protein signatures indicates the source difference or source status of the first and second protein samples. The method further comprises creating a second protein signature from the second protein sample and comparing the first protein signature with the second protein signature, wherein the first and second protein signatures The difference indicates the difference in protein modification between the first and second protein samples.

  In various embodiments of all aspects of the invention, the second protein sample is a first protein sample, except that the cell from which the first protein sample is obtained is a modification defect associated with the cell from which the second protein sample is obtained. It may be a sample obtained from the same type of cells as one protein sample. The second protein sample may be a sample obtained from a different type of cell than the first protein sample, and the cell from which the first protein sample is obtained is a modification associated with the cell from which the second protein sample is obtained. It is deficient. A protein sample can be obtained from one or more cells. Protein signatures can indicate the physiological state of a cell. Protein signatures can show the effect of cell processing. Cells can be obtained from a living body, and cells can be treated by treating the living body. Living organisms can be treated by applying compounds. The living body may be a human.

  In various embodiments of all aspects of the invention, the protein sample can be generated by a separation procedure, which includes liquid chromatography, gel electrophoresis, two-dimensional chromatography, two-dimensional gel electrophoresis, isoelectric focus, thin layer Chromatography, centrifugation, filtration, ion chromatography, mono-affinity chromatography, membrane separation, or combinations thereof can be provided.

  In various embodiments of all aspects of the invention, the set of reporter signal calibrators can consist of a single reporter signal calibrator. The protein signature of the protein sample can represent the presence, absence, amount, or presence and amount of the target protein fragment in the protein sample corresponding to the reporter signal calibrator. Target protein fragments and reporter signal calibrators can be distinguished and / or separated from other molecules based on the common properties of target protein fragments and reporter signal calibrators. The target protein fragment and reporter signal calibrator can be changed after separation. Target protein fragments can be generated by processing a protein sample. In situations where a target protein fragment can be separated from another protein fragment in a protein sample, one or more indicator signal calibrators can generate a predetermined pattern.

  In various embodiments of all aspects of the invention, the method comprises determining a ratio of the amount of target protein fragment detected to the amount of reporter signal calibrator, and the ratio determined and the amount of target protein fragment and reporter. The method further includes the step of comparing the predicted ratio of the amount of the signal calibrator. The prediction ratio can be based on the predicted amount of the target protein fragment in the protein sample and a predetermined amount of the reporter signal calibrator, and if the known amount of protein in the protein sample consists of the target protein (or target protein fragment), the target The predicted amount of the protein fragment is the amount of the target protein fragment protein sample, and the difference between the determination ratio and the prediction ratio is a measure of the purity of the protein sample relative to the target protein (or target protein fragment), and the determined ratio is predicted The closer the ratio, the higher the purity of the protein sample. The reporter signal calibrator and one or more indicator signal calibrators can generate a predetermined pattern. The reporter signal calibrator can generate a predetermined pattern.

  In various embodiments of all aspects of the invention, the method further comprises the step of mixing the target protein fragment and a set of reporter signal calibrators before or simultaneously with step (b), wherein each target protein fragment is Share common characteristics with at least one reporter signal calibrator, and by common characteristics, distinguish and / or separate target protein fragments having common characteristics (eg, each of these and reporter signal calibrator from molecules lacking common characteristics) Reporter signal calibrators (for example, each of these) can be changed, and each type of reporter signal calibrator in the modified format can be modified It can be distinguished from protein fragments.

  According to another aspect, the present invention provides a method of detecting expression, the method comprising detecting a target altered reporter signal peptide obtained from one or more expression samples, wherein the one or more expression samples are A set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, and a reporter signal peptide (or reporter signal peptide) Amino acid segments comprising a signal peptide) have common properties, and the common properties allow the reporter signal peptide (or amino acid segment) to be distinguished and / or separated from molecules lacking common properties, Each modified reporter signal peptide (or amino acid segment) can be distinguished from another modified reporter signal peptide (or amino acid segment) in the modified form, and the target modified reporter signal peptide (or , The target altered amino acid segment) is one of the altered reporter signal peptides (or one of the altered amino acid segments) and the detection of the target altered reporter signal peptide (or target altered amino acid segment) Comprises a nucleotide segment that encodes a reporter signal peptide amino acid segment from which a target altered reporter signal peptide (or target altered amino acid segment) is obtained (or a reporter signal peptide). In a situation where the expression of an amino acid segment comprising a reporter signal peptide (comprising a domain) is shown and the common property allows the reporter signal peptide to be distinguished and / or separated from molecules lacking the common property, Amino acid segment) and / or one or more indicator signal peptides generate a predetermined pattern. In some embodiments, altering the reporter signal peptide alters the amino acid segment.

  According to another aspect, the present invention provides a method for detecting expression, the method comprising detecting altered amino acid subsegments from which one or more expression samples are obtained, comprising: The sample collectively comprises a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, each amino acid segment comprising an amino acid segment. Each amino acid subsegment comprises a part of the protein or peptide and all or part of a reporter signal peptide or indicator signal peptide, and an amino comprising all or part of the reporter signal peptide. Subsegments have common properties that allow amino acid subsegments with all or part of the reporter signal peptide to be distinguished and / or separated from molecules lacking common properties, and the reporter signal peptide can be altered A change in the reporter signal peptide alters the amino acid subsegment, each amino acid subsegment in the altered form is distinguishable from another amino acid subsegment in the altered form, and the target altered amino acid subsegment is altered Detection of the target altered amino acid subsegment indicates expression of the amino acid segment from which the target altered amino acid subsegment is obtained, comprising all or part of the reporter signal peptide, depending on the common characteristics. In situations that can be an amino acid subsegments to distinguish and / or separated from molecules lacking the common property, the amino acid subsegments will generate a predetermined pattern.

  According to another aspect, the present invention provides a method of detecting expression, the method comprising detecting a target altered reporter signal peptide obtained from one or more expression samples, wherein the one or more expression samples are Collectively, it comprises a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, and two reporter signal peptides (or amino acid segments). It belongs to one of the above reporter signal peptides (or amino acid segments), the reporter signal peptides (or amino acid segments) in each set have common characteristics, and each set of reporter signal peptides (or amino acid segments) Segume G) is different from the common characteristics of another set of reporter signal peptides (or amino acid segments), and the common characteristics distinguish and / or separate reporter signal peptides (or amino acid segments) from molecules lacking common characteristics. The reporter signal peptide can be altered, and each altered reporter signal peptide (or amino acid segment) can be distinguished from another altered reporter signal peptide (or amino acid segment) in the target The altered reporter signal peptide (or target altered amino acid segment) is one of the altered reporter signal peptides (or one of the altered amino acid segments) and the target altered reporter signal. Detection of a peptide (or a target-modified amino acid segment) is performed by detecting a reporter signal peptide (or a nucleotide segment encoding an amino acid segment having a reporter signal peptide) from which a target-modified reporter signal peptide (or target-modified amino acid segment) is obtained In a situation where the reporter signal peptide (or amino acid segment comprising the reporter signal peptide) can be distinguished and / or separated from the molecule lacking the common property by the common property. Alternatively, the amino acid segment) generates a predetermined pattern. In some embodiments, altering the reporter signal peptide alters the amino acid segment.

  According to yet another aspect, the present invention provides a method for detecting expression, the method comprising detecting an altered amino acid subsegment from which one or more expression samples are obtained, comprising: The sample collectively comprises a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, each amino acid segment comprising an amino acid sub-sequence. Each amino acid subsegment comprises a part of the protein or peptide and all or part of a reporter signal peptide, and the amino acid subsegment belongs to one of two or more sets of amino acid subsegments Each set Amino acid sub-segments have common characteristics, and the common characteristics of each set of amino acid sub-segments are different from the common characteristics of another set of amino acid sub-segments, depending on the common characteristics, the amino acids comprising all or part of the reporter signal peptide Subsegments can be distinguished and / or separated from molecules lacking common properties, reporter signal peptides can be altered, reporter signal peptide alterations can alter amino acid subsegments, and each form of amino acid subsegment can be altered Is distinguishable from another amino acid subsegment in the altered form, the target altered amino acid subsegment is one of the altered amino acid subsegments, segment In situations where the expression of the resulting amino acid segment is shown and the common property allows the amino acid subsegment comprising all or part of the reporter signal peptide to be distinguished and / or separated from the molecule lacking the common property, the amino acid subsegment is A predetermined pattern is generated.

  According to a further aspect, the present invention provides a method of detecting a cell or cell sample, the method comprising detecting a target-modified reporter signal peptide obtained from one or more cells or cell samples. The cells or one or more cell samples collectively comprise a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and the protein or peptide. Comprising a nucleotide segment encoding an amino acid segment, the reporter signal peptide has a common property, and the common property can distinguish and / or separate the reporter signal peptide from molecules lacking the common property, Each reporter signal peptide in a modified form is distinguishable from another reporter signal peptide in a modified form, and the target modified reporter signal peptide is one of the modified reporter signal peptides , Detection of the target-change reporter signal peptide indicates the presence of a cell or cell sample from which the target-change reporter signal peptide is obtained, and the common property may distinguish or separate the reporter signal from molecules lacking the common property In kill situations, the reporter signal peptide and one or more indicator signal peptides will generate a predetermined pattern.

  According to another aspect, the present invention provides a method of detecting a cell or cell sample, the method comprising detecting a target-modified reporter signal peptide obtained from one or more cells or cell samples, The above cells or one or more cell samples collectively comprise a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, The reporter signal peptide belongs to one of two or more sets of reporter signal peptides, the reporter signal peptides in each set have common properties, and the common properties of each set of reporter signal peptides are different sets of reporter signals. Unlike the common properties of signal peptides, The general properties can distinguish and / or separate the reporter signal peptide from molecules lacking common properties, the reporter signal peptide can be altered, and each altered reporter signal peptide can have a different The reporter signal peptide is distinguishable from the reporter signal peptide, the target-modified reporter signal peptide is one of the modified reporter signal peptides, and detection of the target-modified reporter signal peptide is performed on a cell or cell sample from which the target-modified reporter signal peptide is obtained. In situations where the presence of a common property allows the reporter signal peptide to be distinguished and / or separated from molecules lacking the common property, the reporter signal peptide produces a predetermined pattern.

  According to another aspect, the invention provides a method for detecting a cell or organism, the method comprising detecting a target-modified reporter signal peptide obtained from one or more cells or organisms, comprising: A cell or one or more organisms collectively comprise a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide. , The reporter signal peptide has common properties, and the common property can distinguish and / or separate the reporter signal peptide from molecules lacking the common property, the reporter signal peptide can be changed, and each of the altered forms Reporter signal peptide changed The target-modified reporter signal peptide is one of the altered reporter signal peptides, and detection of the target-modified reporter signal peptide results in a target-modified reporter signal peptide. In a situation where the reporter signal peptide and one or more indicator signal peptides have a predetermined pattern in a situation where the reporter signal peptide can be distinguished and / or separated from molecules lacking the common property by Generate.

  According to another aspect, the present invention provides a method of detecting a cell or organism, the method comprising detecting a target-modified amino acid segment obtained from one or more cells or organisms, comprising one or more cells Or one or more organisms collectively comprise a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide; Amino acid segments with a reporter signal peptide have common characteristics that allow the amino acid segment with the reporter signal peptide to be distinguished from and / or separated from molecules lacking the common characteristics and the reporter signal peptide can be altered And Altering the target signal peptide alters the amino acid segment, each amino acid segment in the altered form is distinguishable from another amino acid segment in the altered form, and the target altered amino acid segment is one of the altered amino acid segments. Detection of the target-modified amino acid segment is indicative of the presence of the cell or organism from which the target-modified amino acid segment is obtained, distinguishing the amino acid segment comprising the reporter signal peptide from the molecule lacking the common property by the common property and / or Or, in situations where they can be separated, amino acid segments produce a predetermined pattern.

  According to another aspect, the present invention provides a method for detecting a cell or organism, the method comprising detecting an altered amino acid subsegment obtained from one or more cells or organisms, comprising: Cells or one or more organisms collectively comprise a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide. Each amino acid segment comprises an amino acid sub-segment, each amino acid sub-segment comprises one part of the protein or peptide and all or part of the reporter signal peptide, and all or part of the reporter signal peptide. Amino acid subunit Have common properties that allow amino acid subsegments comprising all or part of the reporter signal peptide to be distinguished and / or separated from molecules lacking common properties, and the reporter signal peptide can be altered. Yes, reporter signal peptide alterations alter amino acid subsegments, each amino acid subsegment in the altered form is distinguishable from another amino acid subsegment in the altered form, and the target altered amino acid subsegment is altered One of the amino acid subsegments, detection of the target-modified amino acid subsegment indicates the presence of the cell or organism from which the target-modified amino acid subsegment is obtained and comprises all or part of the reporter signal peptide, depending on the common characteristics Amino In situations that can be distinguished and / or separated subsegment from molecules lacking the common property, the amino acid subsegments will generate a predetermined pattern.

  According to another aspect, the present invention provides a method for detecting a cell or organism, the method comprising detecting a target-modified reporter signal peptide obtained from one or more cells or organisms, comprising: A cell or one or more organisms collectively comprise a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, and a reporter signal peptide Belong to one of two or more sets of reporter signal peptides, the reporter signal peptides in each set have common characteristics, and the common characteristics of each set of reporter signal peptides are that of another set of reporter signal peptides. Unlike common characteristics, the common characteristics The signal peptide can be distinguished and / or separated from molecules lacking common properties, the reporter signal peptide can be altered, and each reporter signal peptide in the altered form can be separated from another reporter signal peptide in the altered form The target-change reporter signal peptide is one of the altered reporter signal peptides, and detection of the target-change reporter signal peptide indicates the presence of the cell or organism from which the target-change reporter signal peptide is obtained, In situations where the properties allow the reporter signal peptide to be distinguished and / or separated from molecules lacking common properties, the reporter signal peptide produces a predetermined pattern.

  According to another aspect, the present invention provides a method of detecting a cell or organism, the method comprising detecting a target-modified amino acid segment obtained from one or more cells or organisms, comprising one or more cells Alternatively, one or more organisms collectively comprise a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, wherein the amino acid segment is 2 Belongs to one of more than one set of amino acid segments, the amino acid segments within each set have common characteristics, and the common characteristics of each set of amino acid segments are different from the common characteristics of another set of amino acid segments The properties distinguish amino acid segments from molecules lacking common properties and / or The reporter signal peptide can be altered, the reporter signal peptide alteration can alter the amino acid segment, and each amino acid segment in the altered form can be distinguished from another amino acid segment in the altered form, The target-modified amino acid segment is one of the modified amino acid segments, and detection of the target-modified amino acid segment indicates the presence of the cell or organism from which the target-modified amino acid segment is obtained. In situations where they can be distinguished and / or separated from the lacking molecule, the amino acid segments produce a predetermined pattern.

  According to another aspect, the invention provides a method of detecting a cell or organism, the method comprising detecting an altered amino acid subsegment obtained from one or more cells or organisms. Or one or more organisms collectively comprise a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, Each comprising an amino acid subsegment, each amino acid subsegment comprising a portion of the protein or peptide and all or a portion of a reporter signal peptide, wherein the amino acid subsegment is one of two or more sets of amino acid subsegments. Amino in each set The sub-segments have common characteristics, the common characteristics of each set of amino acid sub-segments are different from the common characteristics of another set of amino acid sub-segments, depending on the common characteristics, the amino acid sub-segment comprising all or part of the reporter signal peptide Can be distinguished and / or separated from molecules lacking common properties, reporter signal peptides can be altered, reporter signal peptide alterations alter amino acid subsegments, and each amino acid subsegment in the altered form is altered The target-modified amino acid subsegment is one of the altered amino acid subsegments, and detection of the target-modified amino acid subsegment is performed when the target-modified amino acid subsegment is can get In situations where the presence of a cell or organism indicates that the common property allows the amino acid subsegment comprising all or part of the reporter signal peptide to be distinguished and / or separated from the molecule lacking the common property, the amino acid subsegment is defined as Generate a pattern.

  In various embodiments of all aspects of the invention, the method further comprises determining the amount of the target altered reporter signal peptide detected. The amount of the target-change reporter signal peptide indicates the amount present in one or more expression samples of the amino acid segment comprising the reporter signal peptide from which the target-change reporter signal peptide is obtained. The amount of amino acid segment present is proportional to the amount of target-change reporter signal peptide detected.

  In various embodiments of all aspects of the invention, the method further comprises the step of determining a plurality of altered reporter signal peptides, wherein detection of each altered reporter signal peptide comprises: The expression of the amino acid segment comprising the resulting reporter signal peptide is shown. The method further comprises determining the amount of altered reporter signal peptide detected, wherein the amount of each altered reporter signal peptide comprises a reporter signal peptide from which the altered reporter signal peptide is obtained. The amount of amino acid segment present in one or more expression samples is indicated. The amount of amino acid segment present is proportional to the amount of altered reporter signal peptide detected.

  In various embodiments of all aspects of the invention, the presence, absence, amount, or presence and amount of the altered form of the reporter signal peptide is such that the altered form of the reporter signal peptide in the expression sample from which the reporter signal peptide is obtained. The presence, absence, amount, or presence and amount of the peptide can be indicated, and the presence, absence, amount, or presence and amount of the reporter signal peptide in the expression sample calibrates the protein signature of the expression sample. Modified forms of reporter signal peptides can be detected by utilizing mass spectrometry, such as using a tandem mass spectrometer. The mass spectrometer can include a quadrupole set for single ion filtering, collision cells, and time-of-flight spectrometers.

  In various embodiments of all aspects of the invention, the reporter signal peptide can be altered by fragmentation or cleavage with a photolabile amino acid. Reporter signal peptides can be fragmented in collision cells and / or fragmented with asparagine-proline bonds, methionine, or phosphorylated amino acids. The common property may be a mass to charge ratio, the reporter signal peptide can be altered by changing its mass, its charge, or its mass and charge, and the altered form of the reporter signal peptide has been altered Differentiation is possible through the mass-to-charge ratio of the reporter signal peptide in the form. The method is 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more. 80 or more, 90 or more, or 100 or more different reporter signal peptides can be used. Ten or more different reporter signal peptides can be used. Each peptide can have unstable or cleavable bonds at various positions.

  In various embodiments of all aspects of the invention, the method further comprises comparing the protein signature with one or more other protein signatures. The detected altered reporter signal peptide can be obtained from multiple expression samples. A portion of the altered reporter signal peptide detected can be obtained from a control expression sample. The method further comprises the step of identifying differences between protein signatures created from the expression sample and the control expression sample. The difference may be a difference in the presence, amount, presence and amount, or absence of reporter signal peptide in the expression sample and the control expression sample. The plurality of expression samples can comprise a control expression sample and a test expression sample such that the test expression sample or the source of the test expression sample destroys, discards, or removes one or more amino acid segments in the test expression sample. The reporter signal peptide corresponding to the amino acid segment that can be processed, destroyed, discarded, or removed is generated from the control expression sample rather than the test expression sample.

  In various embodiments of all aspects of the invention, the test expression sample can be processed to destroy, discard, or remove one or more amino acid segments in the test expression sample. One or more amino acid segments in the test sample can be removed by separating one or more amino acid segments from the test expression sample. One or more amino acid segments can be separated by affinity separation. The source of the test expression sample can be processed to destroy, discard, or remove one or more amino acid segments in the test expression sample. The processing of the source can be performed by exposing the cells from which the test sample is obtained to a compound, composition, or situation that reduces or eliminates the expression of one or more nucleotide segments.

  In various embodiments of all aspects of the invention, the method further comprises identifying a difference in reporter signal peptide in the control expression sample and the test expression sample. The method further comprises identifying a difference between reporter signal peptides in the expression sample. The at least two expression samples or the source of the at least two expression samples may be placed in different situations. The source of the expression sample may be a cell. Differences in protein signatures of at least two expression samples can indicate the effects of different situations. Different situations can be affected by different compounds. Different situations may or may not be affected by the compound.

  In various embodiments of all aspects of the invention, the method further comprises the steps of creating a second protein signature from the second expression sample and comparing the first protein signature to the second protein signature. The difference between the first and second protein signatures indicates the difference in the source or source status of the first and second expression samples. The method further comprises creating a second protein signature from the second expression sample, and comparing the first protein signature with the second protein signature, the first and second protein signatures. The difference in indicates the difference in protein modification between the first and second expression samples. The second expression sample is a sample obtained from the same type of cells as the first expression sample, except that the cell from which the first expression sample is obtained is a modification deficiency associated with the cell from which the second expression sample is obtained. It may be. The second expression sample may be a sample obtained from a different type of cell than the first expression sample, and the cell from which the first protein sample is obtained relates to the cell from which the second protein sample is obtained. Modification deficiency.

  In various embodiments of all aspects of the invention, an expression sample can be obtained from one or more cells. The protein signature can indicate the physiological state of the cell or can indicate the effect of processing the cell. Cells can be obtained from living organisms, and cells can be treated by treating living organisms. A living body can be treated by administering the compound to the living body. The living body may be a human.

  It will be appreciated that in various embodiments of all aspects of the invention, the altered reporter signal peptide can be detected in the first and second expression samples. The second expression sample may be a sample obtained from the same living body, a different living body, a different kind of living body, a different kind of living body, or the same kind of living body as the first expression sample. The second expression sample may be a sample obtained from the same type of tissue as the first expression sample. The second expression sample can be obtained at a different time than the first expression sample. The second expression sample may be a sample obtained from a different type of tissue, or a sample obtained from a different cell compartment than the first expression sample.

  In various embodiments of all aspects of the invention, the method further comprises modifying the reporter signal peptide. The reporter signal peptide can be altered by fragmentation or cleavage with a photolabile amino acid. The reporter signal peptide can be fragmented in the collision cell. Reporter signal peptides can be fragmented with asparagine-proline bonds, methionine, or phosphorylated amino acids.

  In various embodiments of all aspects of the invention, the method further comprises separating the reporter signal peptide from the expression sample. Reporter signal peptides can be distinguished and / or separated from expression samples based on common properties. The method further comprises cleaving the reporter signal peptide from the protein or peptide. Reporter signal peptides can be distinguished and / or separated from the protein or peptide based on common properties. The method further comprises cleaving the amino acid segment into a reporter signal peptide portion and a protein portion. The method further comprises mixing two or more expression samples together.

  In various embodiments of all aspects of the invention, the method further comprises mixing two or more amino acid segments together, wherein the mixed amino acid segments are obtained from two or more different expression samples. Expression of an amino acid segment comprising a reporter signal peptide that yields a target-change reporter signal peptide can identify an expression sample from which the target-change reporter signal peptide is obtained. An expression sample can be obtained from one or more cells, and the expression of the amino acid segment comprising the reporter signal peptide from which the targeted altered reporter signal peptide is obtained identifies the cell from which the identified expression sample is obtained. An expression sample can be obtained from one or more organisms, and the expression of the amino acid segment comprising the reporter signal peptide from which the target-change reporter signal peptide is obtained identifies the organism from which the identified expression sample is obtained. An expression sample can be obtained from one or more tissues, and the expression of the amino acid segment comprising the reporter signal peptide from which the targeted altered reporter signal peptide is obtained identifies the tissue from which the identified expression sample is obtained.

  In various embodiments of all aspects of the invention, expression samples can be obtained from one or more cell lines, and the expression of the amino acid segment comprising the reporter signal peptide from which the altered target reporter signal peptide is obtained has been identified. Identify the cell line from which the expression sample is obtained. Each nucleic acid molecule can further comprise an expression sequence, and the expression sequence can be operably linked to the nucleotide segment such that the amino acid segment is expressed. The expression sequence can comprise a translational expression sequence and / or a transcriptional expression sequence. The amino acid segment can be expressed in vitro or in vivo. Amino acid segments can be expressed in cell culture. The expression sequence of each nucleic acid molecule may be different. Different expression sequences can be differentially adjusted. The expression sequence can be similarly adjusted.

  In various embodiments of all aspects of the invention, the plurality of expression sequences may be or may be derived from genes expressed as part of the same expression cascade. Is understood. The expression sequence of each nucleic acid molecule may be the same or may be adjusted similarly. The expression sequences of at least two nucleic acid molecules may be different or the same. Expression of the amino acid segment can be induced. Each nucleic acid molecule can further comprise a replication sequence, which mediates replication of the nucleic acid molecule. Nucleic acid molecules can be replicated in vitro or in vivo. Nucleic acid molecules can be replicated in cell culture. Each nucleic acid molecule can further comprise an integration sequence, which mediates the integration of the nucleic acid molecule into other nucleic acids. The nucleic acid molecule can be integrated into the chromosome (eg, at a predetermined location).

  In various embodiments of all aspects of the invention, nucleic acid molecules can be created by replicating nucleic acids in one or more nucleic acid samples. Nucleic acids can be replicated utilizing a pair of primers, and each first primer within a primer pair used to create a nucleic acid molecule comprises a nucleotide sequence that encodes a reporter signal peptide. Each first primer can further comprise an expression sequence. The nucleotide sequence of each first primer can also encode an epitope tag. Each amino acid segment can further comprise an epitope tag. The epitope tag of each amino acid segment may be different or the same. The epitope tags of at least two amino acid segments may be different or the same. Amino acid segments can be distinguished and / or separated from one or more expression samples via an epitope tag.

  In various embodiments of all aspects of the invention, the reporter signal peptide of each amino acid segment may be different or the same. The reporter signal peptides of at least two amino acid segments may be different or the same. The nucleic acid molecule can be in a cell or cell line. Each nucleic acid molecule can be in a different cell (or cell line) or in the same cell (or cell line). Each nucleic acid molecule can further comprise an expression sequence, and the expression sequence can be operably linked to the nucleotide segment such that the amino acid segment is expressed. The expression sequence of each nucleic acid molecule may be different or may be similarly adjusted. The plurality of expression sequences may be expression sequences of genes expressed as part of the same expression cascade, or may be obtained from the genes.

  In various embodiments of all aspects of the invention, the nucleic acid molecule can be integrated into the chromosome of a cell (or cell line). The nucleic acid molecule can be integrated at a predetermined position of the chromosome. The chromosome may be an artificial chromosome. The nucleic acid molecule may be a plasmid or may be incorporated into a plasmid. The cell may be in a cell line. Each nucleic acid molecule may be in a different cell or cell line or in the same cell or cell line. Expression samples may be made from cells. Each expression sample can be made from cells obtained from a cell sample, and each expression sample can be made from various cell samples. Each cell sample can be placed in a variety of situations, can be contacted with a variety of test compounds, can be cultured under a variety of circumstances, can be obtained from a variety of organisms, and can be obtained from a variety of tissues. Or can be obtained from the same source at various times. An expression sample can be prepared by lysing cells.

  In various embodiments of all aspects of the invention, the nucleic acid molecule may be in vivo. Each nucleic acid molecule may be in a different living body or in the same living body. Each nucleic acid molecule can further comprise an expression sequence, and the expression sequence can be operably linked to the nucleotide segment so that the amino acid segment can be expressed. The expression sequence of each nucleic acid molecule may be different or may be adjusted similarly. The plurality of expression sequences may be expression sequences of genes expressed as part of the same expression cascade, or may be obtained from the genes. The nucleic acid molecule can be incorporated into a chromosome of a living body (for example, incorporated into a chromosome at a predetermined position). The chromosome may be an artificial chromosome. The nucleic acid molecule may be a plasmid or may be incorporated into a plasmid. Each nucleic acid molecule may be in a different living body or in the same living body. The nucleic acid molecule may be in a living cell (eg, in nearly all of the living cell or in a portion of the living cell). The amino acid segment can be expressed in almost all cells of a living body or a portion of cells of a living body.

  In various embodiments of all aspects of the invention, the protein or peptide of each amino acid segment may be different or the same. The protein or peptide of the at least two amino acid segments may be different or the same. The protein or peptide may be related, may be a protein produced in the same cascade, may be a protein in the same enzymatic pathway, or is a protein expressed in the same context. It may be a protein associated with the same disease or a protein associated with the same cell type or the same tissue type.

  In various embodiments of all aspects of the invention, the nucleotide segment may encode a plurality of amino acid segments each comprising a reporter signal peptide or indicator signal peptide and the protein or peptide. The protein or peptide of at least two amino acid segments within one of the nucleotide segments may be different. The protein or peptide of the amino acid segment within one of the nucleotide segments may be different. The protein or peptide of at least two amino acid segments within each nucleotide segment may be different. The protein or peptide of the amino acid segment within each nucleotide segment may be different.

  In various embodiments of all aspects of the invention, the set can consist of a single nucleic acid molecule. The set can be composed of a single nucleic acid molecule, wherein the nucleic acid molecule comprises a plurality of nucleotide segments, each encoding an amino acid segment. The amino acid segment can comprise a cleavage site near the junction between the reporter signal peptide and the protein or peptide. The cleavage site can be cleaved. The reporter signal peptide can be distinguished and / or separated from the peptide or protein. The cleavage site may be a trypsin cleavage site. The cleavage site may be at the junction between the reporter signal peptide and the protein or peptide. Each amino acid segment can further comprise a self-cleaving segment. The self-cleaving segment may be between the reporter signal peptide and the protein or peptide. Self-cleaving segments can cleave amino acid segments. The reporter signal peptide can be distinguished and / or separated from the peptide or protein. The self-cleaving segment may be an intei segment.

  In various embodiments of all aspects of the invention, a plurality of different altered reporter signal peptides can be detected, and detection of each altered reporter signal peptide results in a reporter signal from which the altered reporter signal peptide is obtained. It is understood that either the expression of the amino acid segment comprising the peptide or the presence of a cell sample from which an altered reporter signal peptide is obtained is shown. Different expression samples or cell samples can comprise different nucleic acid molecules and detection of each altered reporter signal peptide encodes an amino acid segment comprising the reporter signal peptide from which the altered reporter signal peptide is obtained Either the expression of an expression sample comprising a nucleic acid molecule comprising a segment or the presence of a cell sample comprising a nucleic acid molecule comprising a nucleotide segment encoding an amino acid segment comprising a reporter signal peptide resulting in an altered reporter signal peptide Indicate.

  In various embodiments of all aspects of the invention, a plurality of different expression samples can be used, each different expression sample comprising a different nucleic acid molecule, and detection of the altered reporter signal peptide was detected, It is understood that expression of an expression sample comprising a nucleic acid molecule comprising a nucleotide segment encoding an amino acid segment comprising a reporter signal peptide is obtained, resulting in an altered reporter signal peptide.

  In various embodiments of all aspects of the invention, each cell or organism can be designed to contain at least one nucleic acid molecule, and the amino acid segment encoded by the nucleotide segment of the nucleic acid molecule in each cell or organism The reporter signal peptides may be different. Each cell having the trait can have the same reporter signal peptide, and a living body having the trait can have the same reporter signal peptide. The trait may be a heterologous gene or a transgene. The heterologous gene or transgene can comprise a nucleic acid molecule. A heterologous gene or transgene can encode an amino acid segment. Multiple different altered reporter signal peptides can be detected, and detection of each altered reporter signal peptide indicates the presence of a cell from which the altered reporter signal peptide is obtained.

  In various embodiments of all aspects of the invention, different cells or organisms can be provided with different nucleic acid molecules, and detection of each altered reporter signal peptide is a reporter from which the altered reporter signal peptide is obtained. It is understood that the presence of a cell or organism comprising a nucleic acid molecule comprising a nucleotide segment that encodes an amino acid segment comprising a signal peptide.

  In various embodiments of all aspects of the invention, a plurality of different cells, cell samples, or organisms can be used, each different cell, cell sample, or organism comprising different nucleic acid molecules, Detection of an altered reporter signal peptide comprises detecting a cell, a cell sample comprising a nucleic acid molecule comprising a nucleotide segment encoding an amino acid segment comprising a reporter signal peptide that results in an altered reporter signal peptide, or It is understood that it indicates the presence of a living body.

  According to another aspect, the invention provides a method for detecting an analyte, the method comprising associating one or more detectors with one or more target samples, each detector comprising a specific binding molecule, a carrier And a group of blocks, the group of blocks comprising a plurality of blocks, the plurality of blocks comprising a set of reporter signals and one or more indicator signals (and / or two or more sets of reporter signals); And detecting a block group. The reporter signals in each set have common characteristics, which can distinguish or separate the reporter signals from molecules lacking common characteristics, the reporter signals can be altered, and each reporter signal in the altered form is It can be distinguished from another modified form of reporter signal. In situations where the common property allows the reporter signal to be distinguished and / or separated from molecules lacking the common property, the reporter signal and one or more indicator signals (or two or more sets of reporter signals) generate a predetermined pattern. In some formats, indicator signals do not have common characteristics. The common property may be the mass to charge ratio, the reporter signal can be altered by changing its mass, and the altered form of the reporter signal is mediated by the difference in mass to charge ratio of the altered form of the reporter signal. Are distinguishable. The mass of the reporter signal can be altered by fragmentation. Changing the reporter signal can also change the charge.

  In various embodiments of all aspects of the invention, a detector can be associated with and detected by one or more analytes. The detection can include, for example, (a) separating a set of reporter signals and one or more indicator signals (and / or two or more sets of reporter signals) each having a common property from molecules lacking the common property. And (b) identifying a predetermined pattern generated by the reporter signal and one or more indicator signals (and / or generated by two or more sets of reporter signals), and (c) generating the predetermined pattern And (d) detecting and distinguishing the altered form of the reporter signal from each other.

  Accordingly, an object of the present invention is to provide a method for multiple determination of the presence, amount, or presence and amount of an analyte. Another object of the present invention is to provide a labeled protein so that the presence, amount, or presence and amount of the protein can be determined. Another object of the present invention is to provide a method for labeling a protein to allow multiple determinations of the presence, amount, or presence and amount of the protein. Another object of the present invention is to provide a method for multiple determination of the presence, amount, or presence and amount of a protein. An object of the present invention is to provide a mass tag signature detection method. An object of the present invention is to provide a method for detecting a protein signature. Another object of the present invention is to provide an assessment of the uniqueness and purity of peptides comprising protein signatures. Another object of the present invention is to provide a method for detecting phosphorylated peptides or other post-translational protein modifications in peptides with protein signatures. Another object of the present invention is to provide a mass tag signature generation kit. Another object of the present invention is to provide a kit for generating protein signatures.

  Additional advantages of the disclosed methods and configurations will be set forth in part in the following description and will be understood in part from the description, or may be learned from practice of the disclosed methods and configurations. The advantages of the disclosed methods and arrangements will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing summary and the following detailed description are exemplary only and are not intended to limit the claimed invention.

  The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate some embodiments of the disclosed methods and configurations, and together with the description, illustrate the principles of the disclosed methods and configurations Useful.

  Current technology is limited to the ability to multiplex labels. In contrast, the disclosed method of the present invention allows for simultaneous readout of many samples and high internal accuracy compared to a sequential readout system. The disclosed methods have advantageous properties that can be used as detection systems in numerous fields such as antibody or protein microarrays, DNA microarrays, expression profiling, comparative genomics, immunology, diagnostic assays, quality control, and the like.

  The disclosed methods and structures can be more readily understood with reference to the following detailed description of specific embodiments, examples contained therein, the drawings, and the foregoing and following description.

  Disclosed are compositions and methods for sensitive detection of one or more analytes (including proteins). In general, the method involves the use of special labeling components called multidimensional signals (MDS). In the disclosed method, the analysis of the multidimensional signal can result in an additional level of analysis or whether it should be done and / or which part of the analyzed substance can be analyzed with an additional level of analysis. Or one or more predetermined patterns that serve to indicate what should be analyzed. This is beneficial because multiple levels of analysis can be time consuming and generate large amounts of data, and the use of a predetermined pattern at the analysis level to indicate which part should be based on further analysis limits the amount of work. The focus is on data collection for the substance. An effective example of the analysis is mass spectrometry, and an effective example of the predetermined pattern is a pattern of a mass analysis peak based on the mass-to-charge ratio.

  Analysis of the isobaric reporter signal by mass spectrometry has been modified in a different format than the first mass analysis, which generally selects substances of a given mass to charge ratio (corresponding to the mass to charge ratio of the isobaric reporter signal). Two mass analyzes are required for the second mass analysis to detect and identify the reporter signal. For samples containing a large number of different analytes labeled with a reporter signal, each different analyte is selected separately in the first mass analysis, and the altered reporter signal is detected separately for each analyte. There is a need. This is very time consuming. This produces a large amount of data even if separation and detection can be achieved simultaneously for multiple labeled analytes. This is because the portion of the sample that collectively covers the entire range of mass to charge ratios needs to undergo a second mass analysis separately to identify the existing reporter signal and associate it with the various analytes. The disclosed method solves this problem by providing a means to identify which samples and which sample parts are to be further analyzed in the next level of analysis.

  The preferred form of the disclosed method uses a combination of isobaric and non-isobaric techniques, resulting in a system with advanced workflow characteristics. In this workflow, mass spectrometric readout enables efficient data collection by scanning non-isobaric labels in the MS dimension (indicator level analysis) that triggers MS / MS events on isobaric labels (reporter signal level analysis). Provide a system.

  The disclosed methods are described in i-PROT (described in US application 2003/0194717, US application 2004/0220412, US application 2003/0124595, US Pat. No. 6,824,981), iTRAQ (US Appropriate isobaric labeling systems such as application 2004/0220412 and PCT application WO 2004/070352), TMT (described in US application 2003/0194717) can be utilized. The isobaric system disclosed herein is an example that provides enhanced data quality through multiplexed MS / MS readout and attributes that do not increase the complexity of the MS spectrum. Such isobaric labeling systems can be combined or multiplexed using the principles disclosed herein to create non-isobaric relationships between isobaric labeling systems. Alternatively, or in addition, the disclosed methods can be performed using suitable non-isobaric labels, such as ICAT labels (described in PCT application WO 00/011208, examples of use of ICAT labels are described in PCT application WO 02/090929 and US applications. No. 2002/0192720), mass defect tags (described in US Application No. 2002/0172961 and Hall et al., J. Mass Spectrometry 38: 809-816 (2003)), US Application No. 2004/0018565. 2003/0100018, 2003/0050453, 2004/0023274, 2002/014673, 2003/0022225, US Pat. Nos. 6,312,893, 6,312,904, 6,629,040, Geysen et al. Other labels, such as those described in Chemistry & Biology 3 (8): 679-688 (1996) can be utilized, and the non-isobaric pressure disclosed herein is an example. When added to the specimen, non-isobaric labels can be distinguished by MS, but isobaric labels can be MS / MS or higher (depending on the level of isobaric label superposition and analysis method). Cost. That is, the isobaric MS species can be decomposed by MS / MS, and the isobaric MS / MS species can be decomposed by MS / MS / MS. The time required to collect the higher order spectrum is generally longer than the lower order spectrum. The disclosed method utilizes the lower order spectrum to trigger more expensive higher order data collection (and thus uses more modest and efficient higher order data collection). In addition, the amount of data storage increases rapidly in the higher order spectrum, and such a trigger system allows the storage of only data relating to the species. Moreover, when only relevant data passes, downstream data mining may be more efficient.

  The disclosed multi-level analysis serves as an indicator that another level or dimension of analysis is feasible and / or an indicator that a portion of the analysis sample should be analyzed at the next level of analysis An analysis level capable of generating one or more predetermined patterns that can serve as an indicator can be referred to as an indicator level, an indicator analysis, or an indicator level analysis. Some forms of multi-level analysis can be performed when one of the analysis levels is an indicator level. Thus, the disclosed indicator level analysis can be combined with another sample and analyte processing or analysis technique or method either before or after indicator analysis.

  A given indicator level analysis need not be an indicator level analysis associated with all existing multidimensional signals. That is, a multidimensional signal that is analyzed or is intended to be actuated or is not intended to be actuated with respect to a predetermined pattern as disclosed herein is within an analysis level with another multidimensional signal that is analyzed with respect to the predetermined pattern. Can exist. The latter analysis results in an analysis of the indicator level associated with the latter multidimensional signal (ie, “another” multidimensional signal). Similarly, analysis of a given reporter signal level need not be an analysis of reporter signal levels associated with all existing multidimensional signals. That is, a multidimensional signal that is intended to be analyzed or acted upon with respect to an identification reporter signal (or another multidimension signal) as disclosed herein is an identification reporter signal (or Can be present within the analysis level with another multidimensional signal (such as a reporter signal) being analyzed with respect to another multidimensional signal). The latter analysis results in an analysis of the reporter signal level associated with the latter multidimension signal (ie, “another” multidimension signal). Furthermore, a given level or round of analysis may be an indicator level analysis associated with some existing multidimensional signal and an analysis of a reporter signal level associated with another existing multidimensional signal.

In some forms of indicator level analysis, a reporter signal having a common characteristic can be used with another multidimensional signal that lacks the common characteristic. This difference can be the basis for the predetermined pattern used in the disclosed method. For example, a set of reporter signals where members within the set have a common property can be used with one or more indicator signals that lack the common property. In another example, a set of reporter signals in which members in the set have a common characteristic is one in which a given other set of members has a common characteristic that is different from the common characteristic of the first set of reporter signals. These can be used with another set of reporter signals. In another example, one or more sets of reporter signals when members of each given set of reporter signals have common characteristics that differ from the common characteristics of members of another set of reporter signals are among multiple sets of reporter signals Can be used with one or more indicator signals that lack one or more of the common characteristics.

  In the disclosed multilevel analysis, an analysis level that includes identification of a reporter signal (or another multidimensional signal) can be referred to as a reporter signal level, a reporter signal identification level, or a reporter signal analysis. Some forms of multi-level analysis can be performed when one of the analysis levels is a reporter signal level. Thus, the disclosed reporter signal level analysis can be combined with other sample and analyte processing or analysis techniques or methods either before or after reporter signal analysis. Some forms of the disclosed methods have an indicator level followed by a reporter signal level. Multiple indicator levels and reporter signal levels can also be combined in the same assay or assay system.

  The relationship of common characteristics can be shown using mass (or mass to charge ratio). As an example, a set of reporter signals in which members of the set have the same mass (common characteristics) can be used with one or more indicator signals that have a different mass than the reporter signal (and thus lack common characteristics). . In another example, a set of reporter signals in which the members of the set have the same mass (common characteristics) is different from the mass (common characteristics) of a given different set of members that is different from the mass (common characteristics) of the first set of reporter signals ( Can be used with one or more other sets of reporter signals having common properties). In another example, one or more sets of reporter signals in which each given set of reporter signal members has a mass (common property) that is different from the mass (common property) of another set of reporter signal members is a plurality of sets of reporter signals. It can be used with one or more indicator signals having a different mass than one or more or all members of the reporter signal. Thus, indicator signals lack the common properties of reporter signals. In these examples, all members of a given set of reporter signals have the same mass (thus making the mass a common property).

  For example, the same relationship can exist for the mass to charge ratio. It should be understood that a mass difference results in a mass-to-charge ratio difference, and where the charge for different species is the same, such mass-to-charge ratio difference is proportional to the mass difference. Thus, references to mass, mass difference, and relative mass should also be considered as references to mass to charge ratio, mass to charge ratio difference, and relative mass to charge ratio. As an example, a set of reporter signals in which members of the set have the same mass to charge ratio (common characteristics) together with one or more indicator signals that have a different mass to charge ratio (and thus lack common characteristics) from the reporter signals Can be used for In another example, a set of reporter signals in which members of the set have the same mass-to-charge ratio (common characteristics), a given other set of members has a mass-to-charge ratio (common characteristics) of the first set of reporter signals. Can be used with one or more other sets of reporter signals having different mass to charge ratios (common properties). In another example, one or more sets of reporter signals in which each given set of reporter signal members has a mass to charge ratio (common characteristic) that is different from the mass to charge ratio (common characteristic) of another set of reporter signal members is , Can be used with one or more indicator signals having different mass to charge ratios with one or more or all members of multiple sets of reporter signals. Thus, indicator signals lack the common properties of reporter signals. In these examples, all members of a given set of reporter signals have the same mass to charge ratio (thus making the mass to charge ratio a common property).

  By using a reporter signal in the disclosed method, a large number of different samples and / or analytes can be efficiently analyzed in the same assay. For example, different reporter signals (belonging to a set of reporter signals if the reporter signals have common properties) can be used to label different sample analytes or to label different analytes. it can. Other multidimensional signals can be used to label analytes of other samples or to label other analytes. For example, different indicator labels (each different from a reporter signal in common properties) can be used to label analytes in other samples. In another example, different reporter signals (which belong to the second set of reporter signals if the reporter signal has a common characteristic different from the common characteristic of the first set of reporter signals) label the analytes of other samples. Can be used for When the first analysis level (indicator level analysis) is performed, the reporter signal differs in common characteristics from another multidimensional signal, and this known difference can be based on a predetermined pattern. In the next level of analysis triggered by a given pattern (reporter signal level analysis), the reporter signal in the portion of the analytical sample indicated by the given pattern can be changed, and the altered form of the reporter signal is detected and distinguished Is done. With each set of reporter signals alone, many samples and / or analytes can be detected differentially, and the use of multiple sets of reporter signals increases the possible multiplex levels. Furthermore, each different set of reporter signals can contribute to the pattern generated by the indicator level analysis, thus making the reporter signal level analysis more efficient.

  Depending on the format of the method, one set of multidimensional signals can be used to analyze analytes in the first sample or first set of samples, or a different set of multidimensional signals can be used in the second sample or second set of samples. Labeling the analyte, mixing the first and second samples to form an analytical sample, and multidimensional signal in the analytical sample—one or more resulting from analyzing the labeled analyte in the multidimensional signal Identifying one or more predetermined patterns, wherein identification of the one or more predetermined patterns is in one or more of the steps of identifying one or more portions of the analysis sample and one or more of the identification portions of the analysis sample Analyzing the multidimensional signal to identify the multidimensional signal present in the identification portion of the analytical sample. Depending on the format of the method, one or both of the multiple sets of multidimensional signals may be a set of reporter signals, and analysis of the multidimensional signals in one or more of the analytical samples of the one or more discriminating moieties Identifies the reporter signal. One or both of multiple sets of multi-dimensional signals can be both reporter signals and indicator signals, one set of reporter signals and indicator signals, reporter signals and one set of indicator signals or one set of reporter signals and one set of indicator signals. Can be included.

  An additional non-limiting form of the disclosed method can include 1-3 steps. A filtering, selection, or separation step that separates an isobaric multidimensional signal (and added analyte or protein) from another molecule that may be present (eg, based on mass-to-charge ratio), fragmenting the multidimensional signal Any fragmentation step that produces fragments with various masses and, for example, based on mass-to-charge ratio, detects multidimensional signals, labeled analytes or proteins, or both, or various multidimensional signals, various Detection step for distinguishing between various labeled analytes, various labeled proteins, or both. Whether the first stage filtering, selection, or separation step should perform a second fragmentation stage and / or what part of the analyzed material can be analyzed at the fragmentation stage or whether it should be analyzed Can be used to create a predetermined pattern. The labeled analyte or labeled protein is preferably shared by the added multidimensional signal, but based on common properties that are not present in most (or preferably all) other molecules present, Distinguished from and / or separated from. The labeled analyte or labeled protein can be distinguished and / or separated from another molecule based on the common properties of the labeled analyte or labeled protein, generally the mass to charge ratio of the labeled analyte or protein. The separated labeled analyte is then processed and / or detected such that different multidimensional signals, different labeled analytes or different labeled proteins, or both, are distinguishable. Various fragments include fragments of a multidimensional signal and a fragmented labeled analyte or protein (consisting of the analyte or protein and the remainder of the multidimensional signal). One or both can be detected, indicating the characteristics of the first labeled analyte. As described below, the method is optimally performed using a tandem mass spectrometer. In the instrument, the isobaric multidimensional signal is first filtered, then the multidimensional signal is fragmented (preferably by collision), and the fragments are distinguished and detected.

  The disclosed method is effective for sensitive detection of one or more analytes. In general, the method includes a multidimensional signal that can be associated with, incorporated into, or otherwise linked to an analyte, or simply used in conjunction with an analyte without significant association between the analyte and the multidimensional signal. Including the use of special labeling components. In some method embodiments, a multidimensional signal (or derivative of a multidimensional signal) is detected to indicate the presence of an associated analyte. In another embodiment, the analyte (or analyte derivative) is detected along with the multidimensional signal (or derivative of the multidimensional signal).

  In some embodiments of the method, multiple sets of multidimensional signals (eg, reporter signals) distinguish multidimensional signals in which two or more multidimensional signals in the set have common characteristics from molecules lacking other common characteristics. And / or can be used if they have one or more common properties that can be separated. In another embodiment, multiple sets of multidimensional signal / analyte conjugates (eg, multiple sets of reporter signal / analyte conjugates) are shared by two or more multidimensional signal / analyte conjugates in a set. Can be used if they have one or more common properties that can be distinguished and / or separated from molecules lacking other common properties. In yet another embodiment, the analyte can be fragmented (before or after binding) to create a multidimensional signal / analyte fragment conjugate (which can be referred to as a fragment conjugate). In such cases, multiple sets of fragment conjugates can allow two or more fragment conjugates in a set to distinguish and / or separate fragment conjugates having common properties from molecules lacking other common properties. It can be used when it has one or more common characteristics. It should be understood that a fragmented specimen is considered a specimen by itself. In view of this, references to fragmented specimens allow reference to both base and fragmented specimens for convenience and clarity when describing a particular implementation.

  A multidimensional signal (eg, a reporter signal or an indicator signal) (within a multidimensional signal and analyte mixture) is present if there is no significant physical relationship between the multidimensional signal and the analyte, or by itself, the absence of the analyte. Can be combined with a specimen. In such cases, if the multidimensional signal is no longer associated with the analyte, multiple sets of multidimensional signals can be derived from molecules in which two or more multidimensional signals in the set have common characteristics from molecules that lack other common characteristics. It can be used if it has one or more common properties that make it possible to distinguish and / or separate dimensional signals.

  In a preferred embodiment, the disclosed method comprises two basic steps: filtering, selecting, or separating to separate a multidimensional signal, labeled analyte, labeled protein, or another molecule that may have a multidimensional signal fusion. And detecting steps for detecting or distinguishing various multi-dimensional signals, various labeled analytes, various labeled proteins, various multi-dimensional signal fusions, or all of them. A multidimensional signal is preferably distinguished and / or separated from another molecule based on common properties that are shared by the multidimensional signal but not present in most (or preferably all) other molecules present. Is done. The separated multidimensional signal is then processed and / or detected such that the various multidimensional signals are distinguishable. The first filtering step (which constitutes indicator level analysis) is to determine whether the second detection step (which constitutes reporter signal level detection) should be performed and / or which part of the analyzed substance in the detection step It can be used to create a predetermined pattern that indicates whether it can be analyzed or should be analyzed. A useful form of the disclosed method involves associating a multidimensional signal with the analyte. Multidimensional signal detection results in detection of the corresponding analyte. Thus, the disclosed method is a common technique for analyte labeling and detection.

  FIG. 1 is a diagram illustrating an example of using multidimensional signals. The two samples are labeled independently of the two sets of multidimensional signals (label set 1 and label set 2). The labeled sample is mixed and subjected to trypsin digestion (which cleaves the protein in the sample). Mixed and trypsinized samples are clarified by HPLC and subjected to two mass analyses. Example 1 provides a test example after the steps shown in FIG.

  A valid example of the logic flow of the disclosed method example is shown in FIG. 4A. Mass spectrometry spectra are collected (first frame) and the spectra are analyzed to detect non-isobaric patterns (second frame). The first two panes correspond to indicator level analysis. The spectrum can be scanned for a predetermined pattern (first circle). If the predetermined pattern is not detected, the indicator level analysis is repeated for another sample or part of the sample (loop from the first circle to the first frame). If a predetermined pattern is detected, the sample portion where the pattern is detected is sent to another level of analysis (first circle; down arrow). A tandem mass spectrometry spectrum is collected in a portion of the sample (third frame) and the spectrum is analyzed to obtain information about the sample. The third and fourth frames correspond to analysis of reporter signal levels. The entire analysis can be repeated on additional samples (loop from second circle to first frame).

  Using Example 1 as an example, the spectrum can be scanned for double peaks that are separated by 18 daltons or multiples of 18 daltons. Detection methods performed by computers of this type are known (eg, pro-iCAT software, Applied Biosystems (product number WC026995; jsp? hierarchyID = 101 & category1st = 1111395 & category2nd = 1111635 & category3rd = 112058)). If a predetermined pattern is not detected, the indicator level analysis is repeated for another sample or sample part (circle; left arrow). If a predetermined pattern is detected, the sample portion where the pattern is detected is sent to another level of analysis (circle; downward arrow). A tandem mass spectrometry spectrum is collected on the sample portion (third frame) and the spectrum is analyzed to obtain information about the sample. The last two frames correspond to analysis of reporter signal levels. FIG. 4 shows examples of analyzes that can be included within and between the two mass spectrometry stages shown in FIG. Example 1 presents a usage example of the logic flow shown in FIG.

  For simplicity, a single protein is shown in FIG. This logic flow can be extended to a broader recovery of proteins. Furthermore, the two sample experiments can be expanded simply by parallel processing of sample creation and pooling strategies. As an example, consider the number of “normal” input sample individuals compared to the “processed” sample population shown in FIG. FIG. 5 is an example of the method shown in FIG. 1 in which two different sample sets (control sample and test sample) are labeled with different members of two different sets of multidimensional signals (label set 1 and label set 2). It is. In this example, five different test samples are each labeled with a different label set 2 member and seven different control samples are each labeled with a different label set 1 member. The marker set may be, for example, the marker set shown in Table 3. The correlation between the label set and the control and test samples is for clarity and does not represent a limitation of the method. The labeled sample is mixed and subjected to trypsin digestion (which cleaves the protein in the sample). Mixed and trypsinized samples are clarified by HPLC and subjected to two mass analyses. A preferred type of method mixes label control and test sample across label set 1 and label set 2.

  In this design, observation of the non-isobaric marker pattern triggers a measurement from the input sample population. The number of individuals based on statistical assumptions about the control and test status can be incorporated into the test. For example, the greater the number of measurements, the higher the statistical confidence. A preferred format is to mix the control and test samples across multiple sets of multi-dimensional signals (label sets) to combat when a particular protein is not present in the control or test sample. That is, each set of multidimensional signals can include one or more controls and one or more test samples. Thereby, the deviation between sets can be reduced, removed, or controlled.

  In the disclosed method, there is no specific limitation on the number of non-isobaric elements. In this example, two sets of multi-dimensional signals that are not isobaric to each other are used, thus providing two non-isobaric elements for the assay. However, as the number of non-isobaric elements increases, the MS spectrum becomes more complex. It is preferred that the separation of the distinguished ions is greater than the resolution of the mass spectrometer used for the measurement.

  The non-isobaric and isobaric elements of the system need not be embodied by the same multidimensional signal or multiple sets of multidimensional signals. In the above example, non-isobaricity is provided through the inclusion or exclusion of otherwise heavy glycine in the molecule of the same composition. As an example, the method can be used as a single set of isobaric multidimensional signals (eg, label set 1) and a second multidimensional signal that provides the non-isobaric nature of the method. For example, acetylation of primary amines is known (Wetzel et al., Bioconjugate Chem 1: 114-122 (1990)). Heavy versus light non-isobaric features can be introduced through reaction with acetic anhydride. As an example, a three element non-isobaric system is created by labeling with acetic anhydride (light), deuterated analog of acetic anhydride (heavy), or perfluorinated analog with acetic anhydride (very heavy). can do. In this case, the MS spectrum can be scanned for a pattern corresponding to this set of three non-isobaric labels, and the MS / MS spectrum then determines the difference between the members of these non-isobaric sets.

  The non-isobaric nature of the method can be incorporated by metabolic means. For example, cells growing on heavy or light lysine cultures incorporate these heavy or light residues, respectively. There is no limitation to this method involving more than two labels.

  As noted above, a preferred form of the disclosed method is the filtering of isobaric multidimensional signals (and attached analytes or proteins) from another molecule based on mass-to-charge ratio, fragments having various masses. Includes fragmentation of multidimensional signals to generate and detection of various fragments based on mass to charge ratio. The first stage of filtering creates a predetermined pattern that indicates whether the second fragmentation stage should be performed and / or what portion of the analyzed material can be analyzed at the fragmentation stage or should be analyzed. Can be used for

  The method is optimally performed using a tandem mass spectrometer, as described above. The same sample can be analyzed with or without fragmentation (by operating with or without collision gas) and the results compared to detect mass-to-charge ratio variations Is done. Both unfragmented and fragmented results should provide diagnostic peaks in combination with peaks with or without fragmentation confirming the included multidimensional signal (and analyte). The above distinction is achieved using appropriate multiple sets of isobaric multidimensional signals, allowing large scale multiplexing of analyte detection.

  The disclosed method is particularly suitable for use with a MALDI-QqTOF mass spectrometer. This method enables high-level and very high sensitivity multi-sample detection. A suitable tandem mass spectrometer is described in Loboda et al. "MALDI-QqTOF Mass Spectrometer Design and Performance", 47th ASMS Conference, Dallas, Texas (1999), Loboda et al. Rapid Comm. Mass Spectrom. 14 (12): 1047-1057 (2000), Shevchenko et al. Anal. Chem. 72: 2132-1142 (2000), and Kruchinsky et al. J. et al. Am. Soc. Mass Spectrom. 11 (6): 493-504 (2000). In such an instrument, a sample is ionized at a source (eg, MALDI) to create charged ions. The ionization conditions are preferably such that primary single charge parent ions are created. The first and third quadrupoles Q0 and Q2 are operated in RF-only mode and serve as ion guides for all charged particles, and the second quadrupole Q1 is operated in RF + DC mode and Passes only mass charge (ie, a substantially narrow mass charge range). This quadrupole selects the mass to charge ratio (m / z). The collision cells around Q2 are filled to a suitable pressure with a gas to break the input ions by collision-induced dissociation (usually the collision gas is chemically inert but a reactive gas is intended). . Preferred molecular systems utilize multidimensional signals including cleavable bonds, labile bonds, or combinations thereof, and these bonds are preferentially broken in Q2 collision cells.

  A MALDI source is preferred for the disclosed method to simplify multiplex analysis of samples obtained from heterogeneous environments such as arrays, beads, microelements, tissue samples, and the like. Examples of such devices are described in Qin et al. "A practical ion trap mass spectrometer for peptide analysis by matrix-assisted laser desorption / ionization", Anal. Chem. 68: 1784-1791 (1996). For heterogeneous assays, electrospray ionization (ESI) sources work very well. Electrospray ionization source devices that interact with the LC system are commercially available (eg, PE-SCIEX QSTAR, Micromass Q-TOF). Note that the ESI source is operated to create multi-charged ions, with double-charged ions being the most common ions in the disclosed method. Such double charged ions are known in the art and do not represent a limitation on the disclosed method. TOF analyzers and quadrupole analyzers are preferred detectors over sector analyzers. Tandem in-time ion trap systems such as Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometers can also be used with the disclosed methods.

  A number of elements contribute to the sensitivity of the disclosed method. The filter quadrupole Q1 selects a narrow mass-to-charge ratio, distinguishes it from other mass-charge ions, and significantly reduces the background from unsuitable ions. For example, for a sample containing a 3000 Dalton wide mass charge distribution, a 2 Dalton mass charge transmission window applied to this distribution can improve signal-to-noise by a factor of at least 3000/2 = 1500. Once the parent ion is selected by the quadrupole Q1, fragmentation of the parent ion, preferably a single charged daughter ion, has the advantage of fragmenting the parent ion into multiple daughter ions throughout the system. For example, a parent ion fragmented into 20 daughter ions results in a signal that is on average 20 times stronger than the parent ion. This signal dilution does not occur for a parent to a single daughter system.

  This preferred system used in the disclosed method has a high duty cycle and can itself quickly collect excellent statistics. When a single set of isobaric parents is used, multiplex detection is achieved without scanning the filter quadrupole (however, the above scan is beneficial for a single analysis of a complex protein sample with multiple labeled proteins. is there). The electrospray source can be operated continuously, the MALDI source can be operated at a few kHz, the quadrupole can be operated continuously, and the time-of-flight analyzer can measure the mass at a repetition rate of a few kHz. The entire charge region can be captured. Thus, the entire system can obtain thousands of measurements every second. In terms of throughput, the advantage of the multiple verification time-of-flight analyzer is that the time-of-flight analyzer detects all fragment ions with the same capture without requiring ion scanning (or stepping) with a quadrupole analyzer, The last stage is greater than the advantage of a quadrupole analyzer.

  Instrumental improvements, including the addition of laser ports along the flight path, allow protein crossover in additional laser-open additional fragmentation methods through photochemical and photophysical processes (eg, selective bond cleavage, selective ionization) Become. By using a laser to fragment proteins after the filter stage, an ultra high throughput TOF-TOF instrument (50-100 kHz system) can be used.

The disclosed method is compatible with techniques involving bulk sample cutting, processing, or fragmentation to simplify the sample prior to introduction of the multi-stage detection system into the first stage. The disclosed method is compatible with any desired sample, including untreated extract and divided samples.

Disclosed material types and embodiments Multidimensional molecular labeling In one form of the disclosed method, referred to as multidimensional molecular labeling (MDML), a multidimensional signal is first associated with the analyte to be detected and / or quantified and then dissociated. Detected. The dissociated multidimensional signal is subjected to indicator level analysis and reporter signal level analysis. As an example, a multidimensional signal can be associated with a specific binding molecule that interacts with the analyte. Such combinations are referred to as multidimensional molecules. Specific binding molecules within the multidimensional molecule interact directly with the analyte and associate the multidimensional signal with the analyte. Alternatively, the multidimensional signal can be indirectly associated with the analyte. Regardless of whether the interaction between the multidimensional signal and the specific binding molecule is direct or indirect, the interaction between the specific binding molecule and the analyte can associate the multidimensional signal with the analyte. The method of the present invention can be performed such that the association between the analyte and the multidimensional signal is part of the information obtained when the multidimensional signal is detected. That is, the fact that the multidimensional signal can be dissociated from the specimen for detection does not obscure the information that the detected multidimensional signal has been associated with the specimen. Multidimension signals used and / or detected using various techniques (eg, multidimension signal labeling, reporter signal calibration, and multidimension signal fusion) can be used in MDML and / or with MDML Can be combined.

The disclosed method improves the sensitivity and accuracy of detection of the analyte or protein. A preferred form of the disclosed method utilizes a multi-stage detection system to increase the resolution of detection of molecules having very similar properties. In one example, the method includes at least two stages. The first stage is based on the unique properties of the multidimensional signal (and the added analyte or protein) and its distinction from all other molecules, multidimensional signal, labeled analyte or protein, or multidimensional signal fusion ( That is, filtration or selection that allows passage or selection of a subset of molecules present). Subsequent stages further separate and / or detect the multidimensional signal, labeled analyte or protein, or multidimensional signal fusion filtered in the first stage. The main aspect of this method is that the multiplexed set of multidimensional signal, labeled analyte or protein, or multidimensional signal fusion is selected by a filter, and the added multidimensional signal is then cleaved, degraded, reacted, or Another modification is to achieve the uniqueness and / or amount of fragmented multidimensional signal, fragmented labeled analyte or protein, and / or fragmented multidimensional signal fusion in the next stage. The multidimensional signal corresponds to the detected daughter fragment.
B. Multidimensional signal labeling In another form of the disclosed method, referred to as multidimensional signal labeling (MDSL) or multidimensional signal protein labeling, multidimensional signals are sensitive to one or more analytes or proteins. Used for detection. The method can detect analytes by detecting multidimensional signals, labeled analytes or labeled proteins, or both, or by distinguishing between different multidimensional signals, different labeled analytes or different labeled proteins, or both. Or detecting the protein. In that method, an analyte or protein that is labeled with a multidimensional signal is analyzed using the multidimensional signal to distinguish the labeled analyte or protein (if the analyte or protein is labeled with a multidimensional signal). The multidimensional signal is subjected to indicator level analysis and / or reporter signal level analysis. Multidimensional signal detection results in detection of the corresponding labeled analyte (if the analyte is labeled with a multidimensional signal) or the corresponding labeled protein (if the protein is labeled with a multidimensional signal) . Detection of the labeled analyte or labeled protein results in detection of the corresponding analyte and protein. The detected analyte can be analyzed using known techniques. Thus, the use of a multidimensional signal as a label provides a unique analyte / label composition or unique protein / label composition that can specifically identify the analyte or protein. Thus, multidimensional signal labeling and multidimensional signal protein labeling are common techniques for analyte, protein labeling, detection, and quantification.

  It should be noted that although mentioned above and elsewhere in this specification for the detection of “protein”, the disclosed methods and configurations include proteins, peptides, and fragments of proteins or peptides. Accordingly, references herein to proteins are intended to refer to proteins, peptides, and proteins or peptide fragments, unless the context clearly indicates otherwise.

  In some embodiments, multidimensional signals are designed to be fragmented to yield fragments of similar charge but different mass. This allows each labeled analyte or protein in the set (and / or each multi-dimensional signal or multi-dimensional signal fusion (eg, reporter signal fusion)) to be distinguished by different mass to charge ratios of the multi-dimensional signal fragments. it can. This is possible because unfragmented multidimensional signals in a set are isobaric, but fragments of different multidimensional signals are not isobaric. In the disclosed method, this allows each protein / multidimensional signal combination (or analyte / multidimensional signal combination or multidimensional signal fusion) to be coupled to the mass charge of the protein / multidimensional signal after fragmentation of the multidimensional signal. A distinction can be made by ratio.

  Therefore, the labeled analyte or labeled protein can be fragmented before analysis. Analyte or protein samples to be analyzed can also be fractionated or separated to reduce sample complexity. Fragmentation and fractionation can also be used together in the same assay. The fragmentation and fractionation can simplify and extend the analysis of the specimen.

  The multidimensional signal can bind to or directly associate with the analyte or protein. For example, a multidimensional signal can be linked to an analyte or protein via a reactive group, or a multidimensional molecule (consisting of a specific binding molecule and a multidimensional signal) can be associated with an analyte or protein. The multidimensional signal can be added to the analyte or protein in any way. For example, a multidimensional signal can be covalently attached to a protein through a sulfur-sulfur bond between a cysteine on the protein and a cysteine on the multidimensional signal. Many other chemicals and techniques for binding compounds to an analyte are known and can be used to bind a multidimensional signal to the analyte. For example, conjugation can be performed with thiols, epoxides, nitriles for thiols, NHS esters, isothiocyanates for amines, and alcohols for carboxylic acids. The multidimensional signal can be added to the analyte directly or indirectly, eg, via a linker.

  Multidimension signals, or constructs containing multidimension signals, can also be added or linked to the analyte by ligation. Nucleic acid ligation methods are known (see, eg, Sambrook et al. “Molecular Cloning: Laboratory Manual”, 2nd Edition, 1989, Cold Spring Harbor Laboratory Press, New York). Efficient protein ligation is known (eg, Dawson et al., “Protein synthesis by natural chemical ligation”, Science 266, 776-9 (1994); Hackeng et al., “Multidomain proteins: anticoagulant microproteins. Chemical synthesis and spontaneous folding of S ", Proc. Natl. Acad. Sci. USA 97: 14074-8 (2000); Dawson et al.," Synthesis of natural proteins by chemical ligation "Ann. Rev. Biochem. ~ 960 (2000); U.S. Patent No. 6,184,344; see PCT Publication WO 98/28434).

  Alternatively, the multidimensional signal can be indirectly associated with the analyte. In this mode, an “encoded” molecule that includes a specific binding molecule and an encoding tag can be associated with an analyte (via the specific binding molecule). Alternatively, the encoding tag can be linked to or directly associated with the analyte. The multidimensional signal associated with the decoding tag (the combination is another form of multidimensional molecule) is then associated with the encoding molecule through the interaction between the encoding tag and the decoding tag. An example of an interaction is hybridization where the encoding and decoding tags are complementary nucleic acid sequences. The result is an indirect association of the multidimensional signal with the analyte. This mode allows all of the interactions between the multidimensional signal and the encoded molecule to be chemistry by using the same kind of encoding and decoding tags for all encoding and multidimensional molecules in the set. Has the advantage of being carried out in a similar manner physically and physically.

  Multidimensional signals used in MDSL can generate one or more predetermined patterns in indicator level analysis. If a multidimensional signal is linked to the analyte, the pattern can be generated by a combination of the multidimensional signal and the analyte.

  Multidimensional signals, such as reporter signals, can be fragmented, degraded, reacted, derivatized, or otherwise modified, preferably in a specific manner. This allows the analyte or protein to which the multidimensional signal is added or fused to be labeled analyte or labeled protein and one of the labeled analyte or protein following fragmentation, degradation, reaction, derivatization, or other modification of the multidimension signal. Can be distinguished by correlated detection of two or more products (labeled analyte is analyte / multidimensional signal combination and labeled protein is protein / multidimensional signal combination). Proteins can also be identified by multidimensional signal fusion and by correlated detection of one or more products of the multidimensional signal fusion, following fragmentation, degradation, reaction, derivatization, or other modifications of the multidimensional signal peptide. Changing the multidimensional signal is a unique detectable method that changes the labeled analyte or labeled protein. Together, detection of a unique labeled analyte or protein and a specific product of the labeled analyte or labeled protein can uniquely identify the analyte or protein. Thus, using the disclosed methods and materials, one or more analytes or proteins can be detected individually or together (eg, in multiple assays). Furthermore, one or more analytes or proteins in one or more samples can be detected in a multiplex manner. For example, with respect to mass spectrometry multidimensional signals, the multidimensional signal is fragmented to yield fragments of similar charge but different mass.

  In some embodiments, multidimensional signals such as reporter signals are used in sets where all multidimensional signals in the set have similar properties (eg, similar mass to charge ratios). Similar properties can distinguish and / or separate multidimensional signals from another molecule lacking one or more properties. In some embodiments, multidimensional signals within a set have the same mass to charge ratio (m / z). That is, the multidimensional signal within the set is isobaric. Thereby, a multidimensional signal (or a specimen to which the multidimensional signal is added) can be accurately separated from another molecule based on the mass-to-charge ratio. As a result of filtering, the signal-to-noise ratio (S / N) for the system is greatly increased, allowing for more sensitive and accurate detection. Alternatively, or additionally, the multidimensional signal is used in a set that has similar properties that allow the resulting labeled analyte to distinguish and / or separate the labeled analyte from another molecule that lacks one or more properties. be able to.

  The analyte can be detected in a variety of ways using the disclosed multidimensional signals. For example, both the analyte and the added multidimensional signal can be detected, one or more fragments of the analyte and the added multidimensional signal can be detected together, and a fragment of the multidimensional signal can be detected. Can be combined or combined.

  One non-limiting form of the disclosed method involves correlation detection of multidimensional signals both before and after fragmentation of multidimensional signals. This allows the labeled analyte or protein to be detected and identified through changes in the labeled analyte or protein. That is, the nature of the detected multidimensional signal (non-fragmented versus fragmented) identifies the analyte or protein to be labeled. Where analyte or protein and multidimensional signals are detected by mass to charge ratio, the change in mass to charge ratio between fragmented and non-fragmented samples provides a basis for comparison. The detection of the mass to charge ratio is preferably accomplished by mass spectrometry.

  As an example, the analyte in the sample can be labeled with a multidimensional signal designed as a mass spectrometry label. The labeled analyte can be subjected to mass spectrometry. Peaks corresponding to the analyte / multidimensional signal are detected. Analytes that are labeled with different multidimensional signals in the assay can produce relevant peaks that form a pattern. The pattern may be used to indicate whether further levels of analysis can be or should be performed and / or which parts of the analyzed material can or should be analyzed with further levels of analysis. it can. Fragmentation of the multidimensional signal within the mass spectrometer (preferably within the collision cell) results in the loss of part of the added multidimensional signal, the appearance of a peak corresponding to the lost fragment, or both events It causes the fluctuation of the peak corresponding to the combination. Importantly, the observed variability depends on which multidimensional signal is on the analyte as the various multidimensional signals, by design, create fragments with different mass to charge ratios. The combined event of detection of the parent mass charge (without the collision gas) and mass charge (with the collision gas) corresponding to the loss of fragments from the multidimensional signal is indicative of the labeled analyte. The identity of the specimen can be determined by standard mass spectrometry techniques such as composition analysis.

  The dominant method of the disclosed method is the use of analytes or proteins labeled with multidimensional signals, or multidimensional signal fusions for testing multiple samples (eg, time series tests or other comparative analyzes). Is use. Knowledge of the temporal response of the biological system after perturbation is a very effective process for seeking an understanding of the biological system. In order to follow the temporal response, a sample of a biological system (eg, cells from cell culture, first synchronized and sacrificed mouse) is obtained at a predetermined time after perturbation. Knowledge of the spatial analyte profile (eg, relative position within a tissue site) is a very effective process for seeking an understanding of biological systems. In the disclosed method, a series of samples can each be labeled with a different multidimensional signal from a set of multidimensional signals. Non-limiting multidimensional signals for this purpose are signals that use a differentially distributed mass. In particular, stable isotopes can be used to ensure that members of a set of multidimensional signals behave identically chemically.

  Labeled analytes can be detected using mass spectrometry that allows sensitive discrimination between molecules based on their mass-to-charge ratio. The disclosed multidimensional signals can be used as general labels in myriad labeling and / or detection techniques. Since multidimensional signal fragments are designed to have a wide range of masses, and each mass is individually distinguishable after detection, one or more sets of isobaric multidimensional signals can be used for multiple labeling of many analytes and / or Can be used for detection. Furthermore, one or more isobaric multidimensional signal sets whose sets are not isobaric with each other provides an effective way to generate a predetermined pattern and enhance the multiplexing capability of the disclosed method. A set resulting from an isobaric set of multidimensional signals when the same or the same type of analyte is labeled with a set of isobaric multidimensional signals (eg, by labeling the same analyte in different samples) The labeled specimen is also at the same pressure. Similarly, non-isobaric multidimensional signals that are not isobaric to other sets and multiple sets of multidimensional signals label the same analyte (eg, by labeling the same analyte in different samples). Can be used to do. The result is a labeled analyte that is not isobaric. A pattern of labeled analytes with different masses is generated. By using isobaric and non-isobaric multidimensional signals or a combination of multiple sets of multidimensional signals to label the same analyte in different samples, mass patterns can be generated in indicator level analysis . Fragmentation of multidimensional signals in the analysis of reporter signal levels divides individually detectable labeled proteins with uniquely different masses into a set of labeled analytes.

  The disclosed method can be used in many modes. For example, the disclosed methods can be used to detect a specific analyte or protein (in a specific sample or samples) or multiple analytes or proteins (in a single sample or multiple samples). In each case, the analyte or protein to be detected can be separated from each other from an unlabeled analyte or another molecule that lacks the properties of the labeled analyte to be detected. For example, analytes or proteins in a sample can generally be labeled with a multidimensional signal, and analytes or proteins can be separated based on the characteristics of the analyte or protein. For example, a separated analyte or protein can have a specific mass to charge ratio (separation based on a mass to charge ratio selects both labeled and unlabeled analytes with a selected mass to charge ratio). In another example, all of the labeled analyte or labeled protein can be distinguished and / or separated from unlabeled molecules based on the characteristics of a multidimensional signal, such as an affinity tag. If different affinity tags are used, one labeled analyte can be distinguished and / or separated from another labeled analyte. Multidimensional signal labeling enables specimen profiling and specimen cataloging.

  In one mode of the disclosed method, multiple analytes or proteins in multiple samples are labeled when all analytes or proteins in a given sample are labeled with the same multidimensional signal. That is, the multidimensional signal is used as a general label for the analyte or protein in the sample. However, each sample uses a different multidimensional signal. This allows the samples as a whole to be compared with each other. By supplementally separating or distinguishing different analytes or proteins in a sample, it is possible to easily analyze many analytes or proteins in many samples in a single assay. For example, multiple sample proteins are labeled with a multidimensional signal as described above, and the samples are mixed together. If some or all of the various labeled proteins are separated, for example, by associating the protein with an antibody on the array, the presence and amount of a given protein in each sample will reflect the multidimensional signal present in each array element It can be determined by identifying. If a protein corresponding to a given array element is present in a particular sample, some of the proteins associated with the array element are labeled with a multidimensional signal that is used to label the particular sample . The detection of the multidimensional signal indicates this. This same relationship applies to all other samples. In addition, the amount of multidimensional signal detected can indicate the amount of a given protein in a given sample, and simultaneous quantification of proteins in multiple samples can specifically determine the level of protein in each sample. An accurate comparison can be made.

  Optionally, the selection step may be preceded by a fractionation step in which a subset of analytes, including those that are or will be labeled, are separated from other components in the sample. For example, proteins with SH2 domains can be separated from other proteins in the cell sample prior to the selection step. Although the above steps are not necessary, the selection step can be improved by reducing the number of extra molecules present.

  In preferred embodiments, a multidimensional signal (or reporter signal or indicator signal) is a property that all multidimensional signals (or reporter signal or indicator signal) in the set have similar characteristics (eg, similar mass to charge ratio). Used in a set. Similar properties can distinguish and / or separate multidimensional signals (or reporter signals or indicator signals) from other molecules that lack one or more properties. In some embodiments, multidimensional signals (or reporter signals or indicator signals) within a set have the same mass to charge ratio (m / z). That is, the multidimensional signal (or reporter signal or indicator signal) within the set is isobaric. This allows multidimensional signals (or reporter signals or indicator signals, or proteins to which they are added) to be accurately separated from another molecule based on mass to charge ratio. As a result of filtering, the signal-to-noise ratio (S / N) for the system is greatly increased, allowing for more sensitive and accurate detection. Alternatively, or in addition, a multidimensional signal (or reporter signal or indicator signal) is similar in that the resulting labeled protein can distinguish and / or separate the labeled protein from another molecule that lacks one or more properties. Can be used in a set to have properties.

  Proteins can be detected using multidimensional signals disclosed in various ways. For example, a protein and an added multidimensional signal can be detected together, one or more peptides of the protein and the added multidimensional signal can be detected together, and fragments of the multidimensional signal can be detected together. Can be detected or combined. Suitable detection includes detection of protein / multidimensional signal or peptide / multidimensional signal both before and after fragmentation of multidimensional signal.

  As an example, proteins in a sample can be labeled with a multidimensional signal designed as a mass spectrometry label. The labeled protein is subjected to trypsin digestion followed by mass analysis of the resulting material. A peak corresponding to a trypsin fragment containing a multidimensional signal is detected. Fragmentation of the multidimensional signal in the mass spectrometer (preferably in the collision cell) results in peak fluctuations corresponding to the loss of part of the added multidimensional signal, peaks corresponding to the lost fragments Or the combination of both events. Importantly, the observed variability depends on which multidimensional signal is on the protein, as various multidimensional signals, by design, produce fragments with different mass to charge ratios. The combined event of detection of the parent mass charge (without collision gas) and the mass charge (with collision gas) corresponding to the loss of fragments from the multidimensional signal is indicative of the labeled protein. The combination event can be performed in the same manner as the detection of the phosphorylation site described above. The uniqueness of a trypsin fragment of a protein can be determined by standard mass spectrometry techniques such as compositional analysis and peptide sequences.

  Not all labeled analyte fragments or labeled protein fragments that are made from a protein sample in the disclosed method are unique. One part protein has the same common motif as a different protein, and one part protein fragment or peptide made from a sample is the same even though it is derived from a different protein. For example, several families of related proteins have the common motif or common amino acid sequence. Thus, depending on the embodiment of the disclosed method, the detection of a unique labeled protein may result in the detection of a common part of related proteins. The above results are advantageous when detection of a protein family is desired. Alternatively, the collective detection of related proteins can be avoided by focusing on the detection of unique fragments (ie, non-identical port ions) of proteins within the family. For convenience, as used herein, the detection of the common portion of multiple related proteins may be directed to the detection of a unique protein, labeled protein, or other component, unless the context clearly indicates otherwise. Is intended to include a reference to

  In the disclosed method, each series of samples can be labeled with various multidimensional signals from a set of multidimensional signals. A suitable multidimensional signal for this purpose is to use a differentially distributed mass. In particular, the use of stable isotopes is preferred to ensure that members of a set of multidimensional signals behave chemically identically but are distinguishable. An example of a set of labels is shown in Table 1, where each of the five time points is labeled with one of the five title labels, and the sample mixture can be read simultaneously. The non-fragmented label is SEQ ID NO: 1 and the fragmented label is amino acids 7-12 of SEQ ID NO: 1.

開示された方法では、これらの標識は、等圧標識と一緒に所定パターンを形成する１つ以上の他の多次元標識と組み合わせて使用される。標識タンパク質は好ましくはその質量電荷比に基づき、分子間の高感度な区別を可能とする質量分析を用いて検出される。開示された多次元シグナルは、無数の標識付けおよび／または検出技術において全般的な標識として使用することができる。多次元シグナル断片は広い範囲の質量（あるいは、質量電荷比）を有するように設計され、各質量（あるいは、質量電荷比）は検出後、個々に区別可能であるため、１セットの等圧多次元シグナルは多くのタンパク質の多重標識付けおよび／または検出のために使用することができる。同一の検体、同種の検体、同一のタンパク質、または同種のタンパク質が（たとえば、異なるサンプル内の同一のタンパク質を標識付けすることによって）１セットの等圧多次元シグナルで標識付けされる場合、等圧セットの多次元シグナルから生じるセットの標識検体または標識タンパク質も等圧となる。多次元シグナルの断片化は、セットの標識検体または標識タンパク質を、特有に異なる質量を有する、個々に検出可能な標識検体またはタンパク質に分割する。

In the disclosed method, these labels are used in combination with one or more other multidimensional labels that form a predetermined pattern with the isobaric labels. The labeled protein is preferably detected based on its mass-to-charge ratio, using mass spectrometry that allows sensitive discrimination between molecules. The disclosed multidimensional signals can be used as general labels in myriad labeling and / or detection techniques. Multi-dimensional signal fragments are designed to have a wide range of masses (or mass-to-charge ratios), and each mass (or mass-to-charge ratio) is individually distinguishable after detection, so a set of isobaric Dimensional signals can be used for multiple labeling and / or detection of many proteins. The same analyte, the same analyte, the same protein, or the same protein is labeled with a set of isobaric multidimensional signals (eg, by labeling the same protein in different samples), etc. The set of labeled analytes or labeled proteins resulting from the multidimensional signal of the pressure set is also isobaric. Multidimensional signal fragmentation divides a set of labeled analytes or labeled proteins into individually detectable labeled analytes or proteins with distinctly different masses.

  If the detection provides information about the analyte, protein or peptide sequence or other structure from which it was detected, the method allows for detection of the analyte, protein, peptide, and protein fragment. For example, the mass or mass to charge ratio, amino acid composition, or amino acid sequence of a protein can be determined. A set of analytes, proteins, peptides and / or protein fragments detected in a sample using a particular multidimensional signal creates a unique set of analyte, protein and peptide information. The method allows complex samples of analytes or proteins to be cataloged quickly and easily in a reproducible manner. Also, the disclosed method should create two “signals” for each analyte, protein, peptide, or peptide fragment in the sample: the first labeled analyte or protein and the modified form of labeled analyte or protein. It is. This allows a set of detected analytes, proteins and peptides to be compared and verified.

  A preferred form of the disclosed method involves the detection of labeled analytes or proteins in two or more samples or proteins in the same assay. This allows for simple and consistent detection of differences between analytes or proteins in the sample. Differential detection is achieved by labeling analytes or proteins within each sample that are labeled with various multidimensional signals. Preferably, the various multidimensional signals used for the various samples constitute an isobaric set. Thus, the same labeled analyte or labeled protein in each sample has the same mass to charge ratio as the labeled analyte or labeled protein in the various samples. However, after fragmentation of the multidimensional signal, each fragmented labeled analyte or protein in the various samples has a different mass to charge ratio and can therefore be detected separately. Everything can be detected with the same measurement. This is a huge advantage in both time and data quality. For example, because samples are inspected in a single process, there is no need to correct or standardize the results of various samples inspected in various processes. Since it is measured in the same process, the relative amount of the same analyte in various samples can be accurately compared. There are no differences that cause inconsistencies between samples.

  A preferred use of this multiple sample mode of the disclosed method is time series analysis of samples. Such a time series is useful for detecting changes in a sample or response over time. For example, changes in analyte or protein levels within a cell culture over time after the addition of a test compound are assessed. In this mode, different time point samples are labeled with different multidimensional signals, preferably constituting an isobaric set. Thus, the same labeled analyte or protein at each time point has the same mass to charge ratio as the labeled analyte or protein at different time points. However, after fragmentation of the multidimensional signal, each fragmented labeled analyte or protein at various times has different mass to charge ratios and can therefore be detected separately.

  The disclosed method can also be used to collect catalog information about unknown analytes and proteins. This specimen or protein discovery mode can easily link the presence or pattern of the specimen or protein with the analysis. For example, a labeled analyte or protein sample can be compared to an analyte of one or more other samples. Analytes or proteins that appear in one or several samples but not in other samples can be analyzed using conventional techniques. The target analyte or protein is distinguishable from the others by the disclosed labeling, detection, and quantification. This mode of the method is preferably performed using mass spectrometry.

  In some embodiments, the disclosed methods can quickly and easily catalog complex analyte or protein samples in a reproducible manner. The catalogs can be compared with each other, and similarly, differences between samples can be conveniently detected by a catalog created with different analytes or protein samples. A catalog incorporating a substantial amount of information about an analyte or protein sample can serve as a fingerprint of the sample that can be used for both detection of the relevant analyte or protein sample and comparison of the analyte or protein sample. For example, the presence or uniqueness of a particular organism can be detected by creating a catalog of specimens and / or proteins of the test organism and comparing the resulting catalog with a reference catalog created from a known organism. Changes and differences in analyte and / or protein patterns can also be detected by creating catalogs of analytes or proteins from various cell samples and comparing the catalogs. Comparison of analyte and / or protein catalogs created with the disclosed methods is simplified, for example, by the high resolution provided by mass spectrometry.

  Each labeled analyte or protein processed with the disclosed method results in a signal based on the nature (eg, mass to charge ratio) of the labeled analyte or protein. A complex analyte or protein sample can create a unique pattern of signals. This pattern allows for the unique cataloging of analytes or protein samples and a sensitive and effective comparison of signal patterns created from various analytes or protein samples.

  The presence, amount, presence and amount, or absence of various labeled analytes or various labeled proteins depends on the analyte or protein, based on the presence or absence of a particular analyte or analyte fragment (or protein or protein fragment) in the sample. A signal pattern is formed that provides a signature or fingerprint of the analyte or protein sample. Thus, cataloging this signal pattern (ie, the presence, amount, presence and amount, or absence pattern of labeled analyte or protein) is a particularly relevant embodiment of the disclosed method.

  A catalog is configured or referred to as, for example, a labeled analyte or protein pattern, a labeled analyte or protein presence pattern, a labeled analyte or protein catalog, or a catalog of analytes or proteins in a sample. The information in the catalog preferably takes the form of mass charge information or composition information. The catalog may contain or consist of other information obtained from information generated by the disclosed methods (eg, the identity of the detected analyte or protein), obtained from other sources or Can be combined with the generated information. The nature of the information in the catalog created using the disclosed methods is useful for protein combinations and / or analysis using known bioinformatics systems and methods.

  The above catalog of analyte or protein samples can be compared to a similar catalog from another sample to detect sample similarities and differences (indicating analyte or protein similarities and differences within a sample) . For example, a catalog of a first analyte or protein sample includes a sample from a living organism that is the same species as the first analyte or protein sample, a sample from a tissue that is the same species as the first analyte or protein sample, a first analyte or protein sample Sample from the same organism, sample from the same source but obtained at a different time from the first analyte or protein sample, sample from a different organism from the first analyte or protein sample, first analyte or protein sample A sample from a different type of tissue, a sample from a different species of organism than the first analyte or protein sample, a sample from a different species of organism from the first analyte or protein sample, or a first analyte or protein sample And sump from different types of living organisms It can be compared to the catalog.

  Homogeneous tissue is the same type of tissue, such as liver tissue, muscle tissue, or skin (which can be obtained from the same or different organisms or types of organisms). The same living body refers to the same individual, animal, or cell. For example, two samples collected from a patient are obtained from the same living body. The same source is similar but broader, eg, refers to the same organism, the same tissue from the same organism, the same analyte, or a sample from the same analyte sample. Samples from the same source to be compared can be collected at various times (thus taking into account possible changes during the time detected). This is particularly useful when processing impacts and changes in circumstances are to be evaluated. Samples from the same source that have undergone different treatments can be collected and compared using the disclosed methods. Different organisms refer to different individual organisms such as different patients, different individual animals. Different organisms include the same species of different organisms or different species of organisms. Different types of organisms refer to different species of organisms such as dogs and cats, humans and mice, or E. coli and Salmonella. Different types of tissue refer to different types of tissue, such as liver and kidney, or skin and brain. Different species of organisms refer to organisms with different species names, as the term is understood in the art.

  When comparing analyte or protein catalogs obtained from related samples, the presence of a subset of correlated pairs of labeled analytes or proteins and their altered formats can be identified. The disclosed methods can be used to detect (and determine the characteristics of) the first labeled analyte or protein and the modified form of labeled analyte or protein. This pair of detected analytes or proteins is characterized by the analyte to be labeled and the particular multidimensional signal used (although not necessarily unique).

  Thus, multidimensional signal labeling and labeling of multidimensional signal proteins allows for analyte and protein profiling, new discovery of analytes and proteins, and cataloging of analytes and proteins. The method provides advantageous properties that can be used as analyte and protein analysis, proteome analysis, proteome, protein expression profiling, discovery of new analytes and proteins, functional genomics, and detection and analysis systems for analyte or protein detection. Have.

Multidimensional signals used and / or detected using various techniques (multidimensional molecular labeling, reporter signal calibration, multidimensional signal fusion, etc.) can be used in MDSL and / or in combination with MDSL. .
C. Reporter Signal and Indicator Signal Calibration In another form of method called Reporter Signal Calibration (RSC), a form of reporter signal called Reporter Signal Calibrator is mixed with an analyte or analyte fragment (or protein or protein fragment). The reporter signal calibrator and the sample or sample fragment (or protein or protein fragment) are modified, and the modified form of the reporter signal calibrator and modified form of the sample or sample fragment (or protein or protein fragment) are detected. Is done. Reporter signal calibrators are useful as a basis for assessing the amount of analyte or protein present. That is, a known amount of reporter signal calibrator is added to assess the amount of analyte or protein present, and the amount of detected analyte or analyte fragment (or protein or protein fragment) detected, The amounts of the altered reporter signal calibrator can be compared and these amounts can be calibrated with the added known amount of reporter signal calibrator (and thus the predicted amount of the altered reporter signal calibrator).

  The reporter signal and another multi-dimensional signal (eg, indicator signal calibrator) used therewith can be subjected to indicator level analysis and reporter signal level analysis. When used together, the indicator signal calibrator can form a predetermined pattern with the reporter signal calibrator. For reporter signal calibration, the reporter signal calibrator preferably shares one or more common characteristics with one or more analytes, and the indicator signal calibrator preferably does not share common characteristics. Rather, the indicator signal calibrator serves to generate a pattern having a reporter signal calibrator.

  The disclosed reporter signal calibration method generates with high sensitivity unique protein signatures that are related to the relative abundance of various proteins within a tissue, micro-organism, or other biological sample. In the disclosed method, the state of a cell or tissue can be defined by identifying and measuring the relative concentration of a small but very informative subset of proteins. Such a measurement is known as a protein signature. Protein signatures are useful, for example, in cancer diagnosis, classification, and staging, drug screening, toxicity testing, and the like.

  In some embodiments, each analyte or analyte fragment (or protein or protein fragment) having common characteristics is distinguished and / or separated from molecules lacking other common characteristics (or proteins or protein fragments). Alternatively, a protein or protein fragment) can share one or more common properties with at least one reporter signal calibrator.

  In some embodiments, the reporter signal calibrator and the analyte and analyte fragment (or protein or protein fragment) can be altered, and the altered format of the analyte or analyte fragment (or protein or protein fragment) can be the analyte or analyte fragment. (Or proteins or protein fragments) can be distinguished from modified forms of reporter signal calibrators that share common properties. In some embodiments, different types of different reporter signal calibrators can be distinguished from each other. In some embodiments, different forms of different analytes or analyte fragments (or proteins or protein fragments) can be distinguished from each other.

  In some reporter signal calibration embodiments, the analyte or analyte fragment (or protein or protein fragment) is altered, and the altered reporter signal calibrator and analyte or analyte fragment (or protein or protein fragment) are detected. The In this case, the analyte or analyte fragment (or protein or protein fragment) can be distinguished from a modified form of reporter signal calibrator in which the analyte or analyte fragment shares common properties.

  In some embodiments, the analyte or analyte fragment (or protein or protein fragment) may be a reporter signal or reporter signal fragment. In this case, the reporter signal calibrator serves as a calibrator for the amount of reporter signal detected.

  Note that when reporter signal calibration is used in combination with proteins and peptides, this form of reporter signal calibration is referred to as reporter signal protein calibration. Reporter signal protein calibration is useful, for example, in creating a protein signature of a protein sample. As used herein, a protein signature is the presence, absence, amount, or presence and amount of a set of proteins or protein surrogates.

  Depending on the embodiment of the reporter signal protein calibration, instability, cleavage, or the presence of a cleaved bond in the protein can be detected and exploited. Peptides, proteins, or protein fragments containing the above bonds (in the rest of the description, collectively referred to as protein fragments for convenience) can be altered by fragmentation at the bonds. In this way, reporter signal calibrators that have properties common to protein fragments (eg, mass to charge ratio) can be used, modified forms of reporter signal calibrators and modified forms (ie, fragmentation) of protein fragments Can be detected and distinguished. In this regard, protein fragments share common properties with the matching reporter signal calibrator, but the modified format of the reporter signal calibrator and modified (eg, because the modified format has different properties such as different mass to charge ratios). Different types of protein fragments can be distinguished.

  As an example of reporter signal protein calibration, the protein sample can be digested with a serine protease, preferably trypsin. Digestion produces a complex mixture of protein fragments. Among these protein fragments, there is a subset containing the dipeptide Asp-Pro (about one protein fragment for every 400). This dipeptide sequence reacts uniquely to fragmentation during mass spectrometry, yielding high yields of ions in fragmentation mode. Since the human proteome consists of at least 2,000,000 different tryptic peptides, the number of protein fragments containing Asp-Pro sequences is approximately 5,000. Since some of these may exist as phosphorylated peptides or other modified forms, the numbers can be even higher. This number is high enough to select a subset of fragmentable protein fragments (about 50-100) that are suitable for generating very useful protein signatures. Peptides containing Asp-Pro dipeptide sequences, peptides containing amino acids modified by intracellular phosphorylation, or peptides containing internal methionine are particularly suitable for use in reporter signal calibration. Alternatively, arginine-terminated tryptic peptides can be modified by reaction with acetylacetone (pentane-2,4-dione) to increase the frequency of fragment ions (Dikler et al., J Mass Spectrom 32: 1337-49 ( 1997)). In order to maximize the amount of protein signature information, the selection of a subset of protein fragments can be performed using bioinformatics.

For this form of reporter signal protein calibration, protein digests are mixed with a specially designed set of reporter signal calibrators and then analyzed using tandem mass spectrometry. For tandem in-space instruments (eg, Micromass Q-Tof ^™ ), the first quadrupole environment is used for single ion filtering (with unique fragment-by-fragment mass molecules that can be obtained from sequence data). Defined), followed by a collision stage for ion fragmentation, and finally a peptide fragment (a weak bond such as Asp-Pro, a bond containing a phosphorylated amino acid, or a bond adjacent to an oxidized amino acid such as methionine sulfoxide) A TOF spectroscopic measurement of Smith et al., Free Radical Res. In the second stage, the signal-to-noise ratio of the TOF measurement is much larger than that of the conventional MS measurement. In general, one reporter signal calibrator is used for each protein fragment in a sample used to construct a protein signature (such a protein fragment is referred to as a signature protein fragment) and an unfragmented signature. Unlike the fragment mass of protein fragments, each is designed to fragment with a mass pattern that is easily detectable. The quadrupole filtering environment is then varied in a range of values (eg, 50) in sequence, corresponding to the mass of each protein fragment preselected to have a protein signature (ie, signature protein fragment). The In each filtration mass environment, there are two signals that can be detected by TOF after fragmentation, one set is derived from a trypsin peptide (ie, the first protein fragment) and the other set to the reporter signal calibrator. Correspond. A reporter signal calibrator makes it possible to calculate the relative abundance of each signature protein fragment. These relative abundance ratios determined for a given sample constitute a protein signature. The detection limit of the tandem mass spectrometer in MS / MS mode is very nice, about 500 molecules of peptide. This level of detection corresponds to about 1,000 times that of MALDI-TOF mass spectrometry and should allow the generation of protein signatures from a single cell.

  As will be appreciated, with this form of reporter signal calibration, the availability of sequences throughout the human genome and many other living organisms will identify protein fragments that are unique in the context of all known proteins. Simplify. That is, the sequence information can be used to identify peptides generated with protein signatures and guide selection of a reporter signal calibrator.

Multidimensional signals that are used and / or detected using various techniques (eg, multidimensional molecular labeling, multidimensional signal labeling, and multidimensional signal fusion) are used in RSC and / or in combination with RSC Is possible.
D. Multidimension Signal Fusion In another form of the disclosed method and configuration referred to as multidimension signal fusion (MDSF), a multidimension signal peptide is combined with the protein or peptide of interest within a single amino acid segment and the multidimension signal peptide Multidimensional signal fusions, altered forms of multidimensional signal peptides, and / or altered forms of multidimensional signal fusion can be detected. The above fusion of the protein and peptide with a multidimensional signal peptide can be expressed as a fusion protein or peptide from a nucleic acid molecule that encodes the amino acid segments that make up the fusion. A fusion protein or peptide is referred to herein as a multidimensional signal fusion. Multidimensional signal peptides, a form of multidimensional signal, monitor and detect with high sensitivity the proteins and peptides to which they are fused and the expression of the genes, vectors, expression constructs and nucleic acids that encode them. Make it possible. In particular, multidimensional signal fusion allows for sensitive multiplex detection of the expression of specific proteins and peptides and / or genes, vectors and expression constructs encoding the proteins and peptides. The disclosed multidimensional signal fusions can also be used for any purpose, such as as a source of multidimensional signals for other forms of the disclosed methods and configurations.

  “Multidimensional signal fusion” refers to a protein, peptide, peptide, in which a multidimensional signal peptide is fused (ie, joined by peptide bonds within the same polypeptide chain), unless the context clearly indicates otherwise. Or refers to a fragment of a protein or peptide. Multidimensional signal fusions can be fragmented prior to analysis, for example by protease digestion. The analyzed expression sample can be fractionated or separated to reduce sample complexity. Fragmentation and fractionation can be used together in the same assay. Such fragmentation and fractionation can simplify and extend the analysis of expression.

  Multidimensional signal fusions can be made by expression from a nucleic acid molecule that encodes the fusion. Thus, the disclosed fusions can generally be designed by designing a multidimensional signal peptide and a nucleic acid segment that encodes the amino acid segment comprising the protein or peptide. A given nucleic acid molecule can comprise one or more nucleic acid segments. A given nucleic acid segment can encode one or more amino acid segments. A given amino acid segment can include one or more multidimensional signal peptides and one or more proteins or peptides of interest. The disclosed amino acid segment consists of a single, continuous polypeptide chain. Thus, multiple amino acid segments may be part of the same continuous polypeptide chain, but all components of a given amino acid segment (ie, the multidimensional signal peptide, protein, peptide) are the same continuous polypeptide. A part of the peptide chain.

  Thus, the disclosed methods can be used to express certain proteins, genes, vectors, and / or labeled (ie, associated with or included in a multidimensional signal fusion) expressed sequences. , Cell lines, and organisms can be used. For example, a set of nucleic acid constructs, each encoding a multidimensional signal fusion with various multidimensional signal peptides, can be used to uniquely label a set of cells, cell lines, and / or organisms. Can do. In the disclosed method, processing of samples from any of the labeled sources results in a unique modified form of multidimensional signal peptide (or amino acid segment) for each source that is distinguishable from other modified forms. Or amino acid subsegments). A particular modified form of detection identifies the source from which it originates. As a more specific example, genetically modified food species (eg, round-up resistant maize species) that have been introduced with nucleic acid constructs that encode multidimensional signal fusions are identified by detecting multidimensional signal fusions. can do.

  The disclosed multidimension signal fusions have specific proteins, genes, vectors, and / or labeled expression sequences (ie, associated with or included in multidimension signal fusions). It is also effective in creating cells, cell lines, and organisms. For example, a set of nucleic acid constructs, each encoding a multidimensional signal fusion with various multidimensional signal peptides, can be used to uniquely label a set of cells, cell lines, and / or organisms. Can do. In the disclosed method, processing of samples from any of the labeled sources results in a unique modified form of multidimensional signal peptide (or amino acid segment) for each source that is distinguishable from other modified forms. Or amino acid subsegments). A particular modified form of detection identifies the source from which it originates. As a more specific example, a nucleic acid construct encoding a multidimensional signal fusion is introduced into a genetically modified food species (eg, a round-up resistant corn species) and identified by detecting the multidimensional signal fusion. be able to. Suitable multidimensional signal peptides used in or associated with various genes, proteins, vectors, constructs, cells, cell lines, or living organisms have a differentially distributed mass. It is what you have. In particular, the use of alternative amino acid sequences using the same amino acid composition is preferred.

  Nucleic acid sequences encoding multidimensional signal peptides can be designed into specific structural sequences of genes. This is the usual situation when the gene encoding the protein to be fused contains an intron, but the sequence encoding the multidimensional signal peptide can be divided between the various structural sequences that are joined. The placement of nucleic acid sequences encoding multidimensional signal peptides into specific structural sequences is useful for monitoring and analyzing alternative splicing of RNA. The appearance of a multidimensional signal peptide in the last protein indicates that the structural sequence encoding the multidimensional signal peptide has been joined to the mRNA.

  The disclosed multidimensional signal fusions can also be used to “tag” specific pathways, regulatory cascades, and another set of genes, proteins, vectors, and / or expression sequences. The labeling may be on the same cell, cell line, or in vivo, or a set of cells, cell lines, or the entire organism. In a non-limiting example, the disclosed methods are used to assess the status and / or expression of a particular pathway, regulatory cascade, and another set of genes, proteins, vectors, and / or expressed sequences. You can also. By using multidimensional signal fusions to “label” the above pathways, cascades, etc. in the same cell, cell line, or organism, or a set of cells, cell lines, or the entire organism, pathways, cascades, And other systems can be evaluated in a single assay and / or compared across cells, cell lines, or organisms. For example, a nucleic acid segment encoding a multidimensional signal fusion can be associated with the endogenous expression sequence, the endogenous gene, the foreign expression sequence, the foreign gene, or a combination thereof. Thereafter, the exogenous construct is introduced into the cell or organism. Thus, the expression of genes and / or expression sequences is assessed by detecting multidimensional signal peptides and / or multidimensional signal fusions. Association with an endogenous expression sequence or gene introduces a nucleic acid molecule (encoding a multidimensional signal fusion), for example, for insertion at the site of the expression sequence or gene, or a vector or other nucleic acid construct This can be accomplished by creating ex vivo (including both endogenous expression sequences or genes and nucleic acid segments encoding multidimensional signal fusions) and introducing the construct into the cell or organism. Many other uses and uses are possible, many of which are illustrated in the examples below. The disclosed multidimensional signal fusions can be used, for example, in any situation and for any purpose in which green fluorescent protein and green fluorescent protein fusion are fused. However, the disclosed multidimension signal proteins are more versatile and more beneficial than green fluorescent proteins due to at least the ability to more highly multiplex the disclosed multidimension signal fusions.

  Multidimensional signal peptides can be used for sensitive detection of one or more proteins (ie, proteins to which the multidimensional signal peptide is fused). In the method, a protein fused with a multidimensional signal peptide is analyzed using the multidimensional signal peptide to distinguish multidimensional signal fusions. Detection of the multidimensional signal peptide indicates the presence of the corresponding protein. The detected protein can then be analyzed using known techniques. Multidimensional signal fusion provides a unique protein / label composition that can clearly identify proteins. This is achieved by using a special multidimensional signal peptide as a label.

  According to the present invention, multidimensional signal fusions can be fragmented, such as by protease digestion, before analysis. The analyzed expression sample can be fractionated or separated to reduce sample complexity. Fragmentation and fractionation can be used together in the same assay. The fragmentation and fractionation can simplify and extend the analysis of expression.

  Altering multidimension signals (eg, reporter signal peptides) in multidimension signal fusions can create a variety of altered compositions. One or all of these modified forms can be detected. For example, modified forms of multi-dimensional signal peptides can be detected, modified forms of amino acid segments (including multi-dimensional signal peptides) can be detected, and modified forms of sub-segments of amino acid segments Can be detected, or a combination of these can be detected. When the multidimensional signal peptide is altered by fragmentation, the result is generally that the multidimensional signal peptide fragment and the protein or peptide and part of the multidimensional signal peptide (ie, the part that is not a multidimensional signal peptide fragment) A modified form of amino acid segment containing.

  The protein or peptide can also be fragmented. The result is a subsegment of the amino acid segment. The amino acid subsegment includes a multidimensional signal peptide and a portion of the protein or peptide. When a multidimensional signal peptide within an amino acid subsegment is altered (which may occur before, during, or after fragmentation of the amino acid segment), the result is the altered form of the amino acid subsegment (and the altered Format multidimensional signal peptide). This altered form of the amino acid subsegment can be detected. When a multidimensional signal peptide is altered by fragmentation, the result is generally a fragment of the multidimensional signal peptide and an altered form (ie, a fragment) amino acid subsegment. In this case, the altered form of the amino acid sub-segment is referred to herein as a multidimensional signal fusion fragment, the protein or peptide portion and the multidimensional signal peptide portion (ie, the portion that is not a multidimensional signal peptide fragment). including.

  As with multidimensional signals, generally, multidimensional signal fusions (also referred to as amino acid segments), multidimensional signal fusion fragments (also referred to as subsegments of multidimensional signal fusions), or multidimensional signal peptides are within a set. Of multidimensional signal fusions, multidimensional signal fusion fragments, or multidimensional signal peptides can be used in sets that can have one or more properties that produce a pattern with indicator level analysis. For example, multidimension signal fusions, multidimension signal fusion fragments, or multidimension signal peptides in a set can separate or distinguish multidimension signal fusions, multidimension signal fusion fragments, or multidimension signal peptides from molecules that lack common properties. Can have one or more common characteristics. In the case of multidimensional signal fusion, amino acid segments and amino acid subsegments can be used in sets where the amino acid segments and amino acid subsegments in the set can have one or more properties that generate a pattern. For example, in multidimensional signal fusion, an amino acid segment and an amino acid subsegment can be one or more that allow the amino acid segment and amino acid subsegment in the set to separate or distinguish each amino acid segment and amino acid subsegment from a molecule lacking common properties. Can be used in sets with common characteristics. In general, the components of a multidimensional signal fusion that have characteristics depend on the components to be detected and / or the mode of the method being used.

  A nucleic acid molecule (or segment thereof) that encodes a multidimensional signal fusion is one or more of which a multidimensional signal peptide of a multidimensional signal fusion encoded by a set of nucleic acid molecules produces a pattern in an indicator level analysis Can be used in a set that can have the following characteristics: Similarly, a nucleic acid molecule (or segment thereof) that encodes an amino acid segment is one in which a multidimensional signal peptide within the amino acid segment (or segment) encoded by a set of nucleic acid molecules produces a pattern. It can be used in a set that can have the above characteristics. A nucleic acid molecule (or segment thereof) that encodes an amino acid segment is one or more amino acid segments encoded by a set of nucleic acid molecules that can separate or distinguish amino acid segments from molecules that lack common properties. Can be used in sets that can have properties. Consider multiple sets of nucleic acid molecules, nucleic acid segments, amino acid segments, multidimensional signal peptides, and other relationships between members of the protein.

  A multidimensional signal peptide, such as a reporter signal peptide, can have one or more common properties that allow the multidimensional signal peptides in the set to separate or distinguish the multidimensional signal peptide from molecules lacking common properties, Can be used in sets. In the case of multidimensional signal fusion, an amino acid segment and an amino acid subsegment have one or more common characteristics that allow the amino acid segment and amino acid subsegment to separate or distinguish from an amino acid segment and an amino acid subsegment, respectively, from molecules that lack common characteristics. Can be used in a set that can have. In general, the components of multidimensional signal fusion with common characteristics depend on the components to be detected and / or the mode of method being used.

  Multidimension signal fusions can include other components in addition to the protein and multidimension signal peptide. For example, multidimension signal fusions can include epitope tags (eg, his-tag, myc-tag, flu-tag, or flag-tag peptides) (eg, Brizzard et al. (1994) “New Monoclonal Antibodies and Peptides Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using elution: see Biotechnology 16: 730-735). Epitope tags can serve as tags that can manipulate, sequester, separate, distinguish, bind and / or constrain multidimensional signal fusions. The use of epitope tags and flag peptides is generally known and can be adapted for use in the disclosed multidimensional signal fusions.

  In a preferred embodiment, a nucleic acid comprising a multidimensional signal peptide, a multidimensional signal fusion (or amino acid segment), a nucleic acid segment encoding a multidimensional signal fusion, and / or a nucleic acid segment encoding a multidimensional signal fusion. Molecules in sets where the multi-dimensional signal peptides, multi-dimensional signal fusions, and / or sub-segments of multi-dimensional signal fusions that make up or are present in the set have similar properties (eg, similar mass-to-charge ratios) used. Similar properties can distinguish and / or separate multidimension signals, multidimension signal fusions, or subdimensions of multidimension signal fusions from other molecules that lack one or more properties. Preferably, the multi-dimensional signals, multi-dimensional signal fusions, or sub-segments of a multi-dimensional signal fusion that comprise or are present in the set have the same mass to charge ratio (m / z). That is, the subdimensions of a multidimensional signal, multidimensional signal fusion, or multidimensional signal fusion within a set are isobaric. This makes it possible to accurately separate a multidimensional signal, a multidimensional signal fusion, or a sub-segment of a multidimensional signal fusion from another molecule based on the mass to charge ratio. As a result of filtering, the signal-to-noise ratio (S / N) for the system is greatly increased, allowing for more sensitive and accurate detection.

  Cells, cell lines, organisms, and expression of genes and proteins can be detected using multidimensional signal fusions disclosed in various ways. For example, a protein and an added multidimensional signal peptide can be detected together, one or more peptides of the protein and an added multidimensional signal peptide can be detected together, and a fragment of a multidimensional signal peptide Can be detected. Suitable detection includes detection of multidimensional signal fusion both before and after fragmentation of the multidimensional signal peptide.

  A preferred form of the disclosed method involves correlated detection of multidimensional signal fusion both before and after fragmentation of the multidimensional signal peptide. This allows genes, proteins, vectors, and expression constructs that are “labeled” with a multidimension signal peptide to be detected and identified through multidimension signal fusion and / or changes in the multidimension signal peptide. That is, the nature of the detected multidimensional signal fusion or multidimensional signal peptide (non-fragmented versus fragmented) indicates the gene, protein, vector, or nucleic acid construct from which it is obtained. When multidimensional signal fusions and multidimensional signal peptides are detected by mass to charge ratio, the change in mass to charge ratio between fragmented and non-fragmented samples provides a basis for comparison. The detection of the mass to charge ratio is preferably accomplished by mass spectrometry.

  Fragmentation of the multidimensional signal peptide in the mass spectrometer (preferably in a collision cell) results in peak fluctuations corresponding to the loss of the added multidimensional signal peptide part, corresponding to the lost fragment The appearance of a peak or a combination of both events. Importantly, the observed variability depends on which multidimensional signal is fused to the protein as various multidimensional signals create fragments with different mass to charge ratios in the peptide design. The combined event of detection of the parent mass charge (without collision gas) and mass charge (with collision gas) corresponding to the loss of fragments from the multidimensional signal indicates multidimensional signal fusion (hence multidimensional signal fusion, gene, Shows the expression of the vector, or the construct encoding it).

  Multidimensional signal fusion can be detected using mass spectrometry, which allows for sensitive discrimination between molecules based on their mass-to-charge ratio. Multi-dimensional signal peptide fragments are designed to have a wide range of masses (or mass-to-charge ratios), and each mass (or mass-to-charge ratio) can be individually distinguished after detection, so a set of isobaric Dimensional signal peptides or multidimensional signal fusions can be used for multiple labeling and / or detection of expression in many genes, proteins, vectors, expression constructs, cells, cell lines, and organisms. Furthermore, the use of one or more isobaric multidimensional signal sets where the sets are not isobaric to each other can generate both predetermined patterns and effective means that enhance the multiplexing performance of the disclosed method.

  A set of multidimensions where the same gene, protein, vector, expression construct, cell, cell line, or organism (or the same gene, protein, vector, expression construct, cell, cell line, or organism) is isobaric Labeled with a signal fusion, or (for example, by “labeling” the same gene, protein, vector, expression construct, cell, cell line, or organism in different samples) with an isobaric multidimensional signal peptide When included, the resulting set of multidimensional signal fusions or multidimensional signal peptides is also isobaric. Multidimensional signal peptide fragmentation splits a set of multidimensional signal peptides into individually detectable multidimensional signal fusion fragments and multidimensional signal peptide fragments having uniquely different masses. Similarly, non-isobaric multidimension signals and multiple sets of multidimension signals that are not isobaric with other sets (eg, the same gene, protein, vector, expression construct, cell, cell line, or organism in different samples) The same gene, protein, vector, expression construct, cell, cell line, or organism (or the same gene, protein, vector, expression construct, cell, cell line, or organism) Can be used to do. The result is multiple sets of multidimensional signal fusions or multidimensional signal peptides that are not isobaric. Multidimensional signal fusions or multidimensional signal peptide patterns with varying mass are generated. Use isobaric and non-isobaric multidimensional signals or a combination of multiple sets of multidimensional signals to label the same gene, protein, vector, expression construct, cell, cell line, or organism in different samples The mass pattern in the indicator level analysis can then be generated. Fragmentation of isobaric multidimensional signals in the analysis of reporter signal levels divides a set of multidimensional signal fusions or multidimensional signal peptides into individually detectable labeled proteins with uniquely different masses.

  Using and / or combining with MDSF multidimensional signals that are used and / or detected using various techniques (eg, multidimensional molecular labeling, multidimensional signal labeling, and reporter signal calibrators) it can.

  Some types of methods label an analyte or protein in a first sample or a first set of samples with one or more isobaric multidimensional signals or one or more sets of isobaric multidimensional signals. Labeling an analyte in the second sample or the second set of samples with one or more different multidimensional signals or one or more different sets of multidimensional signals; Mixing two samples to form an analytical sample, and analyzing a multidimensional signal-labeled analyte in the analytical sample to identify one or more predetermined patterns resulting from the multidimensional signal, The identification of the predetermined pattern is the step of identifying one or more parts of the analysis sample and the multidimensional signal of one or more identification parts of the one or more analysis samples. Analyzing the multidimensional signal present in the identification part of the analysis sample, wherein the analysis of the multidimensional signal of one or more identification parts of the one or more analysis samples The steps achieved by fragmentation and detection of various multidimensional signal fragments based on mass-to-charge ratio. Depending on the format of the method, the one or more sets of multidimensional signals may be a set of reporter signals, and analysis of the multidimensional signals in one or more identification portions of one or more analytical samples Identify the reporter signal. One or more sets of multi-dimensional signals include, for example, both a reporter signal and an indicator signal, a set of reporter signals and indicator signals, a reporter signal and a set of indicator signals, or a set of reporter signals and a set of indicators. A signal can be included. For example, as described elsewhere herein, the method can be performed using a tandem mass spectrometer.

  Nucleic acid sequences and segments that fuse multidimensional signal fusions can be expressed in any suitable manner. For example, the disclosed nucleic acid sequences and nucleic acid segments can be expressed in vitro, in cells, and / or in cells of living organisms. Many techniques and systems for the expression of nucleic acid sequences and proteins are known and can be used with the disclosed multidimensional signal fusions. For example, many expression sequences, vector systems, conversion and transfer techniques, and recombinant bioproduction methods are known and can be used with the disclosed multidimensional signal peptide methods and compositions.

  For example, kits for in vitro transcription / translation of DNA constructs containing promoters and nucleic acid sequences to be transcribed and translated are known (eg, PROTEINcrypt-PRO (TM) from Ambion, Inc. Austin TX; Wilkinson (1999). ) "Cell-Free And Happy: In vitro translation and transcription / translation system", The Scientific 13 [13]: 15, Jun. 21, 1999). The constructs can be used in genomic DNA of living organisms, plasmids or other vectors that can be transferred into living organisms, or in vitro systems. For example, constructs containing promoter and nucleic acid sequences are known (eg, Sawin and Nurse, “Green Fluorescent Proteins”) that, after transcription and translation, produce green fluorescent protein or luciferase as a multidimensional / marker in an in vivo system. Identification of Fission Yeast Nuclear Markers Using Random Polypeptide Fusions of Proc. Natl. Acad. Sci. USA 93 (26): 15146-51 (1996); Chatterjee et al., “Nuclear Protein Transport in Mammalian Cells In vivo analysis, Exp Cell Res 236 (l): 346-50 (1997); Patterson et al., "Quantitative image of TATA-binding protein in yeast cells of organisms", Yeast 14 (9): 813-25. (199 8); Dhandyuthapani et al., “Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages”, Mol Microbiol 17 (5): 901-12 (1995); Kremer et al., “Green Fluorescent Protein as a New Expression Marker in Mycobacteria”, Mol Microbiol 17 (5): 913-22 (1995); Reilander et al., “Luminous Owan Jellyfish in Insect Cells Using Baculovirus Expression System” Functional expression of green fluorescent protein ", Biochem Biophys Res Commun 219 (1): 14-20 (1996); Mankertz et al.," Human Occludin Promo. Expression is affected by tumor necrosis factor-α and interferon γ ”, J Cell Sci, 113: 2085-90 (2000); White et al.,“ Transcriptional regulation of HTV and hCMV promoters in a single mammalian cell. Real-time analysis of ", J Cell Sci, 108: 441-55 (1995)). It has been found that the green fluorescent protein or a variant thereof is stably incorporated and does not interfere with the living body. In general, since GFP is larger than the disclosed multidimensional signal peptides (GFP from Luminous Aequorea is 238 amino acids in size; NCBI GL606384), generally relatively small multidimensional signal peptides disrupt the added expression system Less likely. For example, where the multidimension is green fluorescent protein (Patterson et al., “Quantitative image of TATA-binding protein in yeast cells of organisms”, Yeast 14 (9): 813-25 (1998)) or luciferase Techniques for modifying promoter regions so that endogenous promoters are replaced with promoter-multidimensional constructs are known. Transcription factor concentration is followed by monitoring GFP or luciferase. These techniques can be used with the disclosed multidimensional signal fusions and multidimensional signal fusion constructs. Techniques for the targeted knock-in of nucleic acid sequences to the gene, usually under the control of an endogenous promoter, are also known. The above techniques that can be used with the disclosed methods and configurations have been used to introduce multidimensional / markers of transcription and translation of genes into which nucleic acids have been inserted. The same technique can be used to place the disclosed multidimensional signal fusions under the control of endogenous expression sequences. Alternatively, non-targeted knock-ins (techniques therefor are also known; Hobbs et al., “Driven by the human polypeptide chain elongation factor 1 alpha promoter to generate stable mammalian cell lines that express very high levels of recombinant proteins. Biochem Biophys Res Commun, 252: 368-72 (1998); Kershnar et al., “Human from a clonal cell line that conditionally expresses the epitope tag subunit of a multiprotein complex”. Immunoaffinity purification and functional characterization of transcription factor IIH and RNA polymerase II ", J Biol Chem, 273: 34444-53 (1998); Wu and Chiang," Tetracycline regulation And Establishing Stable Cell Lines Expressing Potentially Toxic Proteins by Epitope Tagging ”, Biotechiques, 21: 718-22, 724-5 (1996)) follow up the level or activity of transcription factors. Multidimensional signal peptide fusions associated with the inserted nucleic acid code can directly indicate transcription / translation activity.

  The disclosed multidimensional signal fusions can also be used to detect and analyze protein interactions with other proteins and molecules. For example, interactive ion traps for protein-protein interactions include the well-known yeast two-hybrid two-hybrid (Fields and Song, “A Novel Genetic System to Detect Protein-Protein Interactions”, Nature 340: 245. -6 (1989); Uetz et al., "Comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae", Nature 403: 623-7 (2000) and related systems (Ausubel et al., Current Protocols in Molecular Biology, John). Wiley & Sons, Inc., 2001; Van Criekinge and Beyaert, “Yeast Two Hybrids: Latest Technology”, Bi logical Procedures Online, 2 (1), 1999. Incorporation of nucleic acid sequences encoding peptide multidimensional signals can be introduced at the end of these systems, such as the commonly used LacZ selection region (LacZ selection is For example, Sambrook et al., “Molecular Cloning: Laboratory Manual”, 2nd Edition, 1989, Cold Spring Harbor Laboratory Press, New York) A set of integrated sequences (eg, each plasmid). Unambiguously detect many interactions (as many different interactions as the multidimensional signal used) at the same time (by a set of plasmids with multidimensional signal coding sequences and LacZ functionality) Can do.

  Another mode of multidimensional signal fusion is a nucleic acid sequence that encodes a multidimensional signal. The constant (c) region of T cell and B cell receptors can be added to the coding sequence. When the C region is joined to the J region after transcription, a multidimensional signal appears at the T or B cell receptor.

  In another mode of multidimensional signal fusion, referred to as multidimensional signal display, the display of specific antigenic peptides by major histocompatibility (MHC) and non-major histocompatibility molecules can be detected and analyzed. It is known that protein antigens are represented by class I and class II MHC molecules because they are processed by antigens that represent cells, and small peptides, usually 8-12 amino acids, are recognized by T cells. The study of specific T cell / peptide-MHC complexes depends on various labeling requirements (either radioactive or fluorescent) and general dependence on antibody reagents that recognize specific receptors and / or peptide-MHC complexes. Therefore, it is technically troublesome.

  There is a need to further expand our knowledge about antigen processing and antigen display. Multidimensional signals that have been designed for specific protein antigens provide new insights into this process and enable new experimental approaches. For example, consider two viral or bacterial proteins, protein A and protein B that differ by only a few amino acids. It would be beneficial to know if they are processed and displayed against immune cells (eg, T cells) with the same efficiency. Test the presence of different displayed multidimensional signals by designing the multidimensional signal to protein A and protein B to display the antigen that displays the cells, and determine whether the protein is processed and displayed efficiently Can do. The presence of multidimensional signal A (present in protein A) and the absence of multidimensional signal B (present in protein B) indicate that protein A has been processed and protein B has not been processed. Subsequently, the lack of antigen processing of protein B will explain why viruses or bacteria are immune from immune surveillance by the immune system. Antigenic peptides are characterized by tethered residues conserved near both the amino terminus and the carboxy terminus, allowing greater heterogeneity in the middle. This heterogeneous central part is a suitable site for adding a multidimensional signal peptide.

  Multidimensional signal peptides suitable for use in or associated with various genes, proteins, vectors, constructs, cells, cell lines, or living organisms are those that use differential mass distribution. I will. In particular, the use of alternative amino acid sequences using the same amino acid composition is preferred.

  Multidimensional signal fusion can be used to monitor and analyze alternative RNA splicing. The biggest problem in translating genomic information into protein expression is to understand mRNA replacement processing and generation of protein isoforms through the use of alternative exons (Black, “Protein Diversity from Alternative Splicing” : Bioinformatics and the challenge to post-genomic biology ", Cell 103: 367-70 (2000)). There are many examples of the use of alternative pre-mRNA splicing to generate protein isoform diversity, for example under the control of red blood cell discrimination (eg Hou and Comboy, “Alternative pre-mRNA splicing regulation during red blood cell discrimination”, Curr Opin Hematol 8: 74-9 (2001)). Since the structural sequence is only 6 amino acids in a protein with more than 2000 amino acids, detection of complex or conjugated protein isoforms is often a difficult task (eg, Cianci et al., “Brain and Muscle” Expresses a unique alternative transcription of all spectrins ", see Biochem 38: 15721-15730 (1999)).

Exon utilization and processing information can be obtained by inserting a nucleic acid sequence encoding a multidimensional signal into the structural sequence (forming a nucleic acid segment encoding a multidimensional signal fusion). For example, it can be inserted into genomic DNA, a suitable small gene construct, or non-endogenous pre-mRNA introduced into a cell. By using a set of multidimensional signals, all the structural sequences of the translated protein can be read out multiple times at once. The use of small gene constructs or constructs that incorporate short exogenous open reading frame DNA sequences into structural sequences, and the integration of foreign DNA associated with functional intron splice elements is an advanced technology used for the incorporation of multidimensional signals (Eg, Gee et al., “Alternative splicing of proteins 4.1R exon 16: ordered excision of flanking introns ensures selection of appropriate splice sites”, Blood 95: 692-9 (2000); Kikumori et al. al., “Pre-mRNA spliceosome-mediated trans-splicing procrastination: a problem for gene therapy?”, Hum Gene Ther 12: 1429-41 (2001); Malik et al., “The second intron is assembled. "Effects on MFG retroviral vectors", Arch Virol 146: 601-9 (2001); Virts and Raschke, "Role of intron sequences in high level expression from CD45 cDNA constructs", J Biol Chem 276: 19913-20 (2001). See). Knowledge of the detection of multidimensional signals, the amount of multidimensional signals, and which multidimensional signals correlate with which exons provides information on the use of exons and alternative splicing.
E. Lipid Multidimensional Signals The disclosed methods and configurations can also be used to monitor lipid composition, distribution, and processing. Lipids are hydrophobic biomolecules that are highly soluble in organic solvents. Lipids have a variety of biological roles that make them valuable targets for monitoring. As a nutrient source, lipids (along with carbohydrates) constitute an important source of cellular energy and intermediate metabolites required for cell signaling and other processes. Lipids that are processed for energy conversion typically pass through various enzyme pathways and produce many metabolites. An overview of these cycles is available in most modern biochemical texts (see, eg, Stryer, 1995). Monitoring of acyl chain metabolites as metabolized is an important tool for lipid and cell biology studies and clinical detection of biochemical diseases such as medium chain acyl CoA dehydrogenase deficiency (see, for example, Zschocke et al. , "Molecular and functional characteristics of mild MCAD deficiency", Hum Genet 108: 404-8 (2001)). Incorporating a multidimensional signal into a lipid or associating a multidimensional signal with a lipid can improve the method of detecting lipids by, for example, more rapid multiplex detection of processed acyl chain metabolites (eg, , Andresen et al., “Medium chain acyl-CoA dehydrogenase (MCAD) mutations identified by neonatal MS / MS-based predictive screening differ from mutations observed in patients with clinical symptoms: mild MCAD deficiency Identification and Characteristics of Newly Epidemic Mutations ", Am J Hum Genet 68: 1408-18. (2001)).

  In another role, lipids function as the most basic and critical component of all biological membranes. The three main types of membrane lipids are phospholipids, glycolipids, and cholesterol. The most abundant of these are phospholipids obtained from glycerol or sphingosine. Glycerol-based phospholipids typically contain two esterified long chain fatty acids (14-24 carbons) and a phosphorylated alcohol or sugar. Lipids based on sphingosine contain a single fatty acid. Collectively, these lipids contribute to the structure and fluidity of biological membranes. Cyclic changes during the treatment of acidic glycophospholipids, such as inositol phospholipid 4,5 phosphate, in particular, regulate a wide range of cellular processes (eg Cantrell, “Phosphoinositide 3-kinase signaling pathway”, J Cell Sci114: 1439-45 (2001); Payrastre et al., "Phosphoinositides: central presence of cellular signals in time and space," Cell Signal 13: 377-87 (2001)). Thus, by incorporating or associating the acyl chain of the molecule with a multidimensional signal, the multidimensional molecule is subsequently incorporated into an in vitro assay such as that used for enzyme determinations or in vivo assays. Rapid sequestration of these lipids into individual cell compartments (eg, Golgi vs. plasma membrane (eg, Godi et al., “ARF mediates recruitment of PtdIns-4-OH kinase-beta and Stimulates the synthesis of PtdIns (4,5) P2 on the body ", see Nat Cell Biol 1: 280-7 (1999)) and allows processing through metabolism and signal pathways as described above.

  It is known that exogenous lipid labels can be easily incorporated into biological systems, and the disclosed multidimensional signals can also be incorporated into such systems. For example, spin-labeled acyl fatty acids and phospholipids are incorporated into the membranes of phospholipid vesicles and cells (see, for example, Kornberg and McConnell, “Internal and External Transport of Phospholipids within the Vesicular Membrane”, Biochemistry 10: 1111-120 ( 1971); Kornberg and McConnell, “Transverse diffusion of phospholipids in the vesicle membrane”, Proc Natl Acad Sci USA 68: 2564- 8 (1971); Arara et al., “Trypsinized Na studied by spin-labeled EPR, Selectivity of the interaction between K-ATPase and lipid-protein ", Biochim Biophys Acta 1371: 163-7 (1998); Alonso et al.," Studied by spin-label electron paramagnetic resonance. That lipid chains in the stratum corneum dynamics ", Chem Phys Lipids104: 101~11 (2000)).

Triglycerides, or glycosphingolipids or acyl chains of glycolipids, and cholesterol can be synthesized to contain multidimensional signals. An example of the multidimensional signal is a lipid made from a fatty chain having a carboxylic acid with a photolabile bond. Examples of photolabile bonds are described in Glattar and Geise, Org. Lett, 2: 2315-2317 (2000); Guillier et al. Chem. Rev. 100: 2091-2157 (2000); Wierenga, U.S. Pat. No. 4,086,254; and elsewhere herein. A set of multidimensional signals can be created by placing cleavable bonds at various positions within the fatty chain (thus resulting in different mass fragments when the bond is broken). Fatty chains with photolabile bonds constitute multidimensional signals. The synthetic multidimensional molecule can be incorporated into the synthetic triglyceride by, for example, a dehydration reaction. Once formed, a set of these synthetic triglycerides is introduced into the biological system as described above. Multidimensional signals can be recovered from the biological system for detection and quantification, for example, by extraction of lipids into chloroform and release of the multidimensional signal from triglycerides using lipase or hydrolysis reactions.
F. Highly Sensitive Coded Detection System Multidimensional signals such as reporter signals and indicator signals can be used as multiple blocks in the detector system described in US Application Publication US-2003-0124595-A1, the contents of the above publication. Are incorporated herein by reference. The detector system can also be referred to as a high sensitivity coded detection system (SCDS). Multiple sets of multidimensional signals, such as multiple sets of reporter signals, can be used as blocks in SCDS. US Application Publication US-2003-0124595-A1 includes a configuration referred to as a detector based on the use of a carrier with a set of optional molecular tags optimized to facilitate subsequent detection. It describes SCDS. Molecular tags are referred to as multiple blocks, and multiple blocks in a set are referred to as block groups. The carrier is preferably linked to a specific recognition molecule by a covalent bond. A specific recognition molecule is called a specific binding molecule. The detector can be used as a reporter in a biological assay with a recognition molecule linked directly or indirectly. Multiple blocks can be optimized by their chemical composition, for example, so as to be efficiently separated by mass spectrometry. Multiple blocks separated by mass resolution preferably differ in molecular weight due to well-analyzed mass (or mass to charge ratio) differences that allow reliable separation. For separation by mass spectrometry, the carrier can be loaded with a reporter signal if the difference between the mass-to-charge ratios of the altered form of the reporter signal can be used to distinguish and detect the carrier.

  US Application Publication No. US-2003-0124595-A1 encodes a target molecule with a signal and then decodes the encoded signal (using a detector with a block group) to enable multiple tests within a sample in a single assay. The SCDS method for detecting the specimen is described. This encoding / decoding decouples the detection of the target molecule from the chemical and physical properties of the target molecule. In its basic form, the method associates one or more detectors with one or more target samples—the detector comprises a block group of specific binding molecules, carriers, and blocks— And block group detection via detection. The detector associates with the target molecule in the target sample via the specific binding molecule. In general, a detector corresponds to one or more target molecules and a group of blocks corresponds to one or more detectors. Thus, detection of a particular block group indicates the presence of a corresponding detector. Next, the presence of a particular detector indicates the presence of the corresponding target molecule.

  This indirect detection in SCDS decouples the detection of the target molecule from the chemical and physical properties of the target molecule by intervening blocks that can have virtually any chemical and physical properties. . In particular, blocks (and the blocks that they constitute) can have specific characteristics that are useful for detection, and blocks and multiple blocks in a test have a very regular or structured relationship with each other be able to. It is not the characteristics of the target molecules that are relevant (incorporated as they are) at the time of detection, but the characteristics of the block group (which can be freely selected) and multiple blocks.

  Multidimensional signals, reporter signals, indicator signals, multiple sets of multidimensional signals, multiple sets of reporter signals, and multiple sets of indicator signals are indicated herein as blocks, detectors or multiple blocks within a detector group. Can be selected to generate such a predetermined pattern. For example, a set of reporter signals can be used with a set of indicator signals, two sets of reporter signals can be used together, and a set of reporter signals can be used with a single indicator signal. . The detection, analysis, and use of the predetermined patterns described herein are for use in detectors and other SCDS components and in the methods described in US application publication US-2003-0124595-A1. Can be utilized in the detection, analysis, and use of the disclosed multidimensional signals. Detectors, blocks, blocks, unique composition, quantity composition are defined in US application publication US-2003-0124595-A1, the definitions of which are incorporated herein by reference.

  Accordingly, the present invention provides a detector having one or more target samples, each detector comprising a specific binding molecule, a carrier, and a block group, the block group comprising a plurality of blocks, and the block comprising a set of blocks. A reporter signal and one or more indicator signals (and / or two or more sets of reporter signals). Reporter signals within each set can have common characteristics, which can distinguish or separate the reporter signal from molecules lacking common characteristics, the reporter signal can be altered, and each of the altered forms Reporter signals are distinguishable from all other modified forms of reporter signals. The reporter signal and one or more indicator signals (or two or more sets of reporter signals) form a predetermined pattern in a situation where the common properties allow the reporter signal to be distinguished and / or separated from molecules lacking the common properties. In some formats, indicator signals do not have common characteristics. The common property may be the mass to charge ratio, the reporter signal can be altered by changing its mass, and the altered form of the reporter signal is mediated by the difference in mass to charge ratio of the altered form of the reporter signal. Are distinguishable. The mass of the reporter signal can be altered by fragmentation. Changing the reporter signal can also change the charge.

  Although the blocks can have the same amount composition, the blocks need not all have the same amount composition. Multiple detectors can be associated with one or more target samples, and each detector block group can have blocks of different composition. Each block group can have the same number of blocks, but the block groups need not all have the same number of blocks. Each block group can have a different unique composition of blocks. Groups of blocks having the same unique composition of blocks can have different quantity compositions of blocks. Detectors, blocks, blocks, unique composition, and quantity composition are defined in US application publication US-2003-0124595-A1, which is incorporated herein by reference.

Blocks can be detected by MALDI-TOF spectroscopy. The block may be an isobaric block. Multiple detectors can be associated with one or more target samples, and each detector block may be different. All blocks of all detectors can have the same mass to charge ratio. A block can be changed by changing its mass, charge, or both, and the modified form of the block can be distinguished via the mass-to-charge ratio difference of the modified form of the block.
The carrier can be selected from the group consisting of beads, liposomes, microparticles, nanoparticles, and branched polymer structures. The carrier may be a bead. The carrier may be a liposome or microbead. Liposomes may be unilamellar vesicles. Vesicles can have an average diameter of 150-300 nanometers. Liposomes can have an inner diameter of 200 nanometers. The carrier may be a dendrimer. The dendrimer may be a contact polymer selected from the group consisting of DNA, RNA, and PNA. The polymer may be an oligonucleotide that is 20-300 nucleotides in length.

  The specific binding molecule can be selected from the group consisting of antibodies, ligands, binding proteins, receptor proteins, haptens, aptamers, carbohydrates, synthetic polyamides, and oligonucleotides. The specific binding molecule may be a binding protein. The binding protein may be a DNA binding protein. The DNA binding protein can include a motif selected from the group consisting of a zinc finger motif, a leucine zipper motif, and a helix-turn-helix motif.

  The specific binding molecule may be an oligonucleotide. Oligonucleotides can have a length of 10-40 nucleotides or a length of 16-25 nucleotides. The oligonucleotide may be a peptide nucleic acid. The oligonucleotide can form a triple helix with the target sequence. The oligonucleotide can comprise a psoralen derivative that can covalently attach the oligonucleotide to a target sequence.

The specific binding molecule may be an antibody that binds a protein. The blocks may be oligonucleotides, carbohydrates, synthetic polyamides, peptide nucleic acids, antibodies, ligands, proteins, haptens, zinc fingers, aptamers, mass labels, or combinations thereof. The specific binding molecule and the carrier can be covalently bonded. Carriers and blocks can be covalently bonded. The specific binding molecule and the carrier can be covalently bonded. The specific binding molecule can comprise a first oligonucleotide and the carrier can comprise a second oligonucleotide that can hybridize to the first oligonucleotide. The first oligonucleotide can be conjugated to an antibody that binds the protein. Also disclosed is a composition for detecting an analyte comprising a specific binding molecule, a carrier, and a block group, wherein the block group comprises a block, the block comprising a set of reporter signals and one or more indicator signals (and / or Or two or more sets of reporter signals). The reporter signals in the set can have common characteristics, which can distinguish or separate the reporter signal from molecules lacking common characteristics, the reporter signal can be modified, and each reporter in the altered format The signal is distinguishable from all other modified forms of the reporter signal. In situations where the common property allows the reporter signal to be distinguished and / or separated from molecules lacking the common property, the reporter signal and one or more indicator signals (or two or more sets of reporter signals) are in a predetermined pattern. Form. In some formats, indicator signals do not have common characteristics.
G. Rearranged multidimensional signal Another embodiment of the disclosed method and configuration, referred to as rearranged multidimensional signal (rearranged MDS or RMDS), has the occurrence of a specific gene rearrangement event, its protein product, and receptor Enables detection of specific cell populations. RMDS can also track the progression and development of specific receptors and cells or cell populations by monitoring the presence and / or absence of multidimensional signals. The design considerations for rearranged multidimensional signals are similar to those required for multidimensional signal fusion as described elsewhere in this specification.

  Most embodiments of the disclosed methods include intact multidimensional signals that are associated with the analyte in various ways. RMDS utilizes processes such as biological processes to form multidimensional signals by specific rearrangements of multidimensional signal pieces or rearrangements of nucleic acid segments that encode only a portion of the multidimensional signal. One form of RMDS utilizes endogenous biological systems such as the variable-diversity-linked (VDJ) gene rearrangement mechanism present in the mammalian immune system. In this system, non-contiguous short elongated germline DNA (V, D, and J gene fragments) are brought together (recombined) before serving as a transcription template. Gene rearrangements occur in leukocytes such as T and B lymphocytes and are the primary mechanism for generating T cell and B cell antigen receptor diversity. Theoretically, billions of different receptors can be generated. This level of complexity makes it difficult to detect the presence of rare rearrangement events or receptors. PCR-based assays and flow cytometry approaches are currently used to detect receptor diversity. However, the PCR approach is difficult and does not provide information about the state of the expressed protein. The flow cytometry approach has limited multiplexing performance due to the overlap of the emission spectra of the fluorescent probes used.

  If it is desired to test 50-100 T cell or B cell receptors, it is necessary to utilize the same number of antibodies against those receptors, which is virtually impossible. Therefore, there is a real need for a method that can specifically detect specific receptors within a highly complex pool of receptors with high sensitivity. The ability to highly multiplex this approach allows for experimental approaches that are not currently achievable. The disclosed multidimensional signal technology allows large-scale multiplexing of signals for detection.

  As an example of RMDS, transgenic mice can be generated in which a nucleic acid sequence encoding a multidimensional signal is designed in the mouse reproductive system. Methods for doing this are known in the art, and standards for rearranging multidimensional signals, eg, designing yeast or bacterial artificial chromosomes (YAC and BAC), and then using these constructs to generate transgenic mice Use of traditional molecular biology methods.

  As an example of the use of immunoglobulin rearrangement for RMDS, one part of a multidimensional signal can be encoded in the D region and another part of the multidimensional signal can be encoded in the J region. During a rearrangement event that combines the D and J regions that encode a “partial” multidimensional signal, an encoded sequence of “complete” multidimensional signals is generated. After transcription and translation, multidimensional signals are encoded within the protein product. The multidimensional signal can then be detected as described elsewhere in the present invention. In the absence of rearrangement events joining the designed D and J regions, no multidimensional signal is detected. By including a sequence encoding portion of various multidimensional signals having different D and J regions, various different multidimensional signals can be converted by different, diagnostic multidimensional signal rearrangements for each of the different possible rearrangements. Can be generated. This system can also be extended to include, for example, multi-dimensional signals that are divided among three or more gene regions (eg, VDJ, VDDJ, etc.). Multiple rearrangement events create multidimensional signals. In this mode, a combination of rearrangements of multidimensional signal portions produces a number of different multidimensional signals, each characterized by a particular multidimensional signal portion rearranged to form a multidimensional signal.

  Transgenic mice carrying RMDS can address problems that were otherwise very difficult or impossible to address. For example, it can be investigated which particular T and B cell receptors (out of thousands or millions) respond to a particular stimulus or which cell type is present at a particular developmental stage.

Transgenic mice carrying rearranged multidimensional signals can address problems that were otherwise very difficult or impossible to address. For example, it can be investigated which particular T and B cell receptors (out of thousands or millions) respond to a particular stimulus or which cell type is present at a particular developmental stage.
H. Mass Spectrometer The disclosed method utilizes a mass spectrometer for analysis of multidimensional signals, modified forms of multidimensional signals, and various analytes and analyte fragments. Mass spectrometers are generally available and such instruments and their operation are known to those skilled in the art. Fragmentation systems integrated with mass spectrometers are commercially available, and exemplary systems include liquid chromatography (LC) and capillary electrophoresis (CE).
The major components of a mass spectrometer are (a) one or more sources, (b) one or more analyzers and / or cells, and (c) one or more detectors. Source types include electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Analyzers and cell types include quadrupole mass filters, hexapole collision cells, ion cyclotron traps, and time of flight (TOF). Detector types include multi-channel plates (MCP) and ion multipliers. Suitable mass spectrometers for use with the disclosed methods are described by Kruchinsky et al. , "Fast MALDI-ion fast protein identification using ion trap mass spectrometer", 49th ASMS conference summary on mass spectrometry and related topics (27-31 May 2001), The Rockefeller University, New York, New York.

  A mass spectrometer with two or more analyzers / cells is known as a tandem mass spectrometer. There are two types of tandem mass spectrometers, as well as analyzers that are a combination and combination of these types: “tandem in space” and “tandem in time” analyzers. A tandem mass spectrometer in which ions traverse two or more analyzers / cells is known as a tandem in-space mass spectrometer. A tandem in-space analyzer utilizes spatially ordered elements and acts on ions as they pass through each element. Tandem mass spectrometers in which ions remain predominantly in one analyzer / cell are known as tandem in-time mass spectrometers. A tandem in-time mass spectrometer utilizes a temporarily ordered operation on ions as they are contained in space. Complex systems and combinations of these types are known. The ability to select a particular mass-to-charge ratio in a mass analyzer is usually characterized by a resolution (reported as the central mass charge divided by the full width at half maximum of the selected ion). Thus, resolution is an indicator of the narrowness of the ion mass-charge distribution that passes through the analyzer to the detector. Reference to the above resolution is generally pointed out herein by referring to the ability of a mass spectrometer to pass only a narrow range of mass to charge ratios.

  A suitable shaped mass spectrometer for use in the disclosed method is a tandem mass spectrometer, such as a tandem in-space tandem mass spectrometer. As an example of the use of a tandem in-space instrument, an isobaric multidimensional signal can first pass through a filtering quadrupole, the multidimensional signal is fragmented (preferably within a collision cell), and the fragment is time of flight ( It is distinguished and detected at the TOF) stage. In the instrument, a sample is ionized with a source (eg, a MALDI ion source) to create charged ions. As the ionization condition, it is preferable that primary single charge parent ions are created. The first quadrupole Q0 is operated only in radio frequency (RF) mode and serves as an ion guide for all charged particles. The second quadrupole Q1 is operated in RF + DC mode and passes only a narrow range of mass to charge ratios (including the mass to charge ratio of multidimensional signals). This quadrupole selects the mass to charge ratio. The quadrupole Q2 surrounded by the collision cell is operated in an RF only mode and serves as an ion guide. When multi-dimensional signal fragmentation is desired, the collision cells around Q2 are filled to the appropriate pressure with a gas so as to fragment the input ions by collision-induced dissociation. The collision gas is preferably chemically inert, but reactive gases can also be used. A preferred molecular system utilizes multidimensional signals, including cleavage bonds, labile bonds, or combinations thereof, to preferentially fragment these bonds within Q2 collision cells.

MS ^N compliant tandem equipment can be used with the disclosed method. As an example, select a set of molecules using a first stage filter (MS), photodissociate these molecules to produce a set of multi-dimensional signals, and use a second stage (MS / MS) We consider how to select these multidimensional signals, change these multidimensional signals by collision fragmentation, and detect them by time of flight (MS / MS / MS or MS3). Many other combinations are possible and the disclosed method can be adapted for use with the system. For example, extending the multi-dimensional signal fragment to more stages or analysis is also included in the art.

It is to be understood that the disclosed methods and configurations are variable, unless otherwise specified, because they are not limited to specific synthetic methods, specific analytical techniques, or specific reagents. Further, it is to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.
Materials The disclosed materials, compositions, and components can be used for, disclosed in conjunction with, can be used in conjunction with the disclosed methods and configurations, or It is their product. As these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc. of these materials are disclosed, various individual and overall combinations and permutations of these compounds It is understood that specific references to should not be explicitly disclosed and each is specifically intended and described herein. For example, if a multidimensional signal is disclosed and described and a number of modifications that can be made to a large number of molecules containing the multidimensional signal are described, then all possible combinations of multidimensional signals and modifications and Reordering is specifically intended unless clearly indicated otherwise. Thus, when classes of molecules A, B, and C are disclosed, examples of molecules D, E, and F and combined molecules AD are also disclosed. Each is intended individually and in groups, even if not individually listed. Thus, in this example, the combinations A-E, A-F, B-D, B-E, B-F, C-D, CE, and C-F are specifically intended and A, B, And C; D, E, and F; and combinations AD should be considered disclosed from disclosure of examples. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-groups AE, BF, and CE are specifically contemplated, and examples of A, B, and C; D, E, and F; and combinations AD It should be considered disclosed from the disclosure. This idea applies to all aspects of this application including, but not limited to, steps in a method of making and using the disclosed steps. Thus, if there are a variety of additional steps that can be performed, each of these additional steps can be implemented with a particular embodiment or combination of embodiments of the disclosed method, and the above combinations are specifically intended and disclosed. It is understood that it should be considered.
A. Multi-dimensional signal Multi-dimensional signal (MDS) can or should perform additional levels of analysis and / or which part of the analyzed material can be analyzed with additional levels of analysis, or A special labeling component that can generate one or more predetermined patterns that serve to indicate what should be analyzed. Reporter signals and indicator signals are in the form of multidimensional signals. A multidimensional signal is a molecule having at least one characteristic that can distinguish and / or separate a multidimensional signal from another multidimensional signal or another set of multidimensional signals. In general, a multidimensional signal need only be distinguishable and / or separable from another multidimensional signal and / or multiple sets of multidimensional signals present in the same indicator level analysis. Some multidimensional signals, such as reporter signals, should also be distinguishable from different reporter signals within a set of reporter signals in the analysis of reporter signal levels after the reporter signal is altered. Thus, multidimensional signals have two main functions or features in the disclosed manner. That is, between the related differences that can generate patterns in the indicator level analysis and the modified forms of multidimensional signals (typically reporter signals) that can distinguish different multidimensional signals in the reporter signal level analysis. It is a difference.

  As described above, the multidimensional signal may be a reporter signal and an indicator signal. Thus, reporter signals and indicator signals are two types of multidimensional signals. A useful form of the disclosed method can include the use of at least one set of multi-dimensional signals. Reporter signals are molecules that can be preferentially fragmented, degraded, reacted, derivatized, or otherwise modified or altered for detection, as detailed below. An indicator signal is a molecule having at least one characteristic that can distinguish and / or separate an indicator signal from another multidimensional signal, as described in detail below. In general, the indicator signal need only be distinguishable and / or separable from other multidimensional signals present in the same level of analysis. Multidimensional signals, reporter signals and indicator signals can be used in the set both individually and together. Thus, for example, a set of reporter signals can be used with a set of indicator signals, two sets of reporter signals can be used together, and a set of reporter signals can be used with a single indicator signal. Can do.

  A multidimensional signal, such as a reporter signal, can have two main characteristics. First, multidimensional signals can be used in sets where all multidimensional signals in the set have similar characteristics. Similar properties allow multidimensional signals to be distinguished and / or separated from other molecules lacking one or more properties. In some embodiments, multidimensional signals within a set have the same mass to charge ratio (m / z). That is, the multidimensional signal within the set is isobaric. This allows accurate separation of multidimensional signals from other molecules based on mass to charge ratio. As a result of the filtering, the signal-to-noise ratio (S / N) for the system is greatly increased, enabling more sensitive and precise detection. Filtering creates a predetermined pattern from a multidimensional signal that indicates whether the second stage should be performed and / or what part of the analyzed material can be analyzed at the fragmentation stage or should be analyzed at the fragmentation stage It can be used to

  Second, all multidimensional signals in the set are fragmented, degraded, reacted, derivatized, or otherwise modified for detection to distinguish them from the various multidimensional signals in the set. can do. For example, multidimensional signals can be fragmented to yield fragments of the same or similar charge but different mass. This allows each multidimensional signal in the set to be distinguished by different mass to charge ratios of the multidimensional signal fragments. This is possible because unfragmented multidimensional signals in the set are isobaric, but the fragments of the various multidimensional signals are not isobaric. A multidimensional signal detected based on mass to charge ratio and / or detected using a mass spectrometer can be referred to as a mass spectrometer multidimensional signal. Reporter signals are one type of multidimensional signal that can have these characteristics.

  Differential mass distribution of fragments of multidimensional signals such as reporter signals can be achieved in a number of ways. For example, multidimensional signals of the same nominal structure (eg, peptides having the same amino acid sequence) are composed of various distributions of heavy isotopes such as deuterium. All multidimensional signals in the set have the same number of given heavy isotopes, but their distribution varies with the various multidimensional signals. Similarly, multidimensional signals of the same general structure (eg, peptides having the same amino acid sequence) consist of different distribution modifications such as methylation, phosphorylation, sulfation, and the use of seleno-methionine for methionine, etc. can do. All multidimensional signals in the set have the same number of given modifications, but their distribution varies with different multidimensional signals. Multidimensional signals of the same nominal composition (eg, composed of the same amino acids) can be composed of different orders of subunits or components of the multidimensional signal. All multidimensional signals in the set have the same number of subunits or components, but their distribution varies with different multidimensional signals. Multidimensional signals having the same nominal composition (eg, composed of the same amino acids) can be composed of unstable or broken bonds at various positions within the multidimensional signal. All multidimensional signals in the set appear to have the same number and order of subunits or components. When unstable or broken bonds exist between specific subunits or components, the order of the subunits or components in the multidimensional signal is the same except for subunits or components that form unstable or broken bonds It is. Each of these modes can be combined with one or more other modes to generate a differential mass distribution of multi-dimensional signal fragments. For example, different distributions of heavy isotopes can be used in multidimensional signals where unstable or broken bonds are made at different positions. Furthermore, each of these modes can be combined with one or more other modes and / or another multidimensional signal to create a differential mass distribution of the multidimensional signal and multiple sets of reporter signals. Mass patterns that can be detected and used with level analysis can be generated.

  Multidimensional signals, such as reporter signals and indicator signals, can be detected using mass spectrometry that allows sensitive discrimination between molecules based on their mass-to-charge ratio. The disclosed multidimensional signals such as reporter signals and indicator signals can be used as general labels in a myriad of labeling and / or detection techniques. A multi-dimensional signal fragment is designed to have a wide range of masses, and each mass (or mass-to-charge ratio) is individually distinguishable after detection, so a set of isobaric multi-dimensional signals can multiplex many analytes. It can be used for labeling and / or detection. The combination of isobaric and non-isobaric multidimensional signals can generate a mass (or mass to charge ratio) pattern, extending the multiplexing of the method.

  Thus, multidimensional signals can be used in sets. For example, a set of multi-dimensional signals that differ in several properties or characteristics can be used to label different samples and / or analytes. In some forms of multidimensional signals, features are used in the reporter signal and / or another multidimensional signal, or the same assay or assay system, so that a recognizable pattern occurs during analysis of the multidimensional signal It can be selected to be compatible with the characteristics of multiple sets of multidimensional signals. For example, a multidimensional signal or multiple sets of multidimensional signals that have different masses (or mass to charge ratios) than the masses (or mass to charge ratios) of another multidimensional signal and multiple sets of multidimensional signals can be Used to generate mass (or mass to charge ratio) feature patterns in mass spectrometry. Multidimensional signals, reporter signals and indicator signals can be used in sets both individually and together. Thus, for example, a set of reporter signals can be used with a set of indicator signals, two sets of reporter signals can be used together, and a set of reporter signals can be used with a single indicator signal. Can do.

  The disclosed multidimensional signals are preferably used in sets where the members of the set have various mass to charge ratios (m / z), or in which one set of members of the multidimensional signal has the same mass to charge ratio, Members of different sets of the sets have different mass to charge ratios. Thereby, based on the mass-to-charge ratio, multidimensional signals and / or multiple sets of multidimensional signals are easily and sensitively distinguished from each other and from other multidimensional signals and / or multiple sets of multidimensional signals. The multidimensional signal can take any structure that can generate a pattern with another multidimensional signal in the analysis of the disclosed method.

  Suitable multidimensional signals (eg, reporter signals or indicator signals) include subunits such as peptides, oligonucleotides, peptide nucleic acids, oligomers, carbohydrates, polymers, and other natural and synthetic polymers, and combinations thereof. Consists of chains. Most suitable chains are peptides, referred to herein as multidimensional signal peptides (or reporter signal peptides or indicator signal peptides as the case may be). The subunits and subunit chains have a similar relationship to the polymer and mer chains. Mers are connected together to form a polymer. Similarly, subunits are connected together to form subunit chains. Suitable multidimensional signals are composed of strands of similar or related subunits. These are called homochains or homopolymers. For example, nucleic acids are composed of phosphonucleosides and peptides are composed of amino acids.

  Multidimensional signals can also be composed of heterochains or heteropolymers. A hetero chain is a chain or polymer in which the subunits constituting the chain are different types, or in which the mer constituting the polymer is different. For example, the heterochain may be guanosine-alanine consisting of one nucleoside subunit and one amino acid subunit. It is understood that any combination of subunit types can be used with the disclosed compositions, sets, and methods. Molecules with the required properties can be used as multidimensional signals. In general, a multidimensional signal need only be distinguishable and / or separable from another multidimensional signal present at the same level of analysis (eg, indicator level analysis). Multidimensional signals, such as some reporter signals, should be distinguishable from different reporter signals within a set of reporter signals in the analysis of reporter signal levels after alteration of the reporter signal.

  Multidimensional signals are preferably given in a set in which all indicator signals in the set have different physical characteristics and / or sets in a set of sets have different physical characteristics. Members of the same set can have the same physical properties). Different (or distinguishing) characteristics distinguish and / or distinguish multidimensional signals and / or multiple sets of multidimensional signals from other multidimensional signals and / or multiple sets of multidimensional signals that differ in one or more characteristics. Can be separated. As an example, multidimensional signals within a set have the same or different mass to charge ratios (m / z). That is, the multidimensional signal within the set may be isobaric or non-isobaric. In general, within the set, the indicator signal can be non-isobaric and the reporter signal can be isobaric.

  A multidimensional signal can be used in combination with another multidimensional signal. In general, at least two different types of multidimensional signals can be used together in the same assay or assay system. Different types of multidimensional signals used together can generate one or more predetermined patterns during analysis that can serve as indicators that can perform another level or dimension of analysis. Each analysis level can then generate one or more predetermined patterns that can serve as indicators that can perform another level or dimension of analysis. The disclosed method generally includes at least two analysis levels, and the pattern generated at the first analysis level indicates whether the second analysis level should be performed. The pattern generated by the analysis of the multidimensional signal can also be used to indicate whether the portion of the material being analyzed should be analyzed in the next level of analysis. Thus, for example, any different portion or fragment of an analytical sample that is split, separated, or otherwise fragmented is identified for the next level of analysis based on the detection of a predetermined pattern generated by the current level of analysis. Or you can choose.

  The pattern generated by the indicator level analysis is a result of one or more characteristics of the multidimensional signal in the assay. For example, two or more different types of multidimensional signals can be used together in the same assay or assay system that differs in one or more attributes. Different types of multidimensional signals used together can generate one or more predetermined patterns during analysis based on this characteristic difference. For example, different types of multidimensional signals with distinct mass (or mass to charge ratio) differences result in a characteristic predetermined pattern of mass (or mass to charge ratio) when analyzed by mass spectrometry. . More specifically, when one set of multidimensional signal members differs from another set of multidimensional signal members by a specific amount, mass (or mass to charge ratio), the two sets of multidimensional signal members are: Different mass spectrometric peaks are generated based on specific mass (or mass to charge ratio) differences. This means that the same analyte fused or conjugated to different types of multidimensional signals generates different mass spectrometric peaks based on unique mass (or mass to charge ratio) differences, so multidimensional signals are analyzed independently. This is true regardless of whether the multidimensional signal fusion or multidimensional signal / analyte conjugate is analyzed. The characteristic mass (or mass-to-charge ratio) difference can be, for example, a mass difference (or mass-to-charge ratio) in the form of a multidimensional signal, multiple mass differences (or mass-to-charge ratio) in the form of a multidimensional signal, or Any combination of the format of the dimensional signal and the mass difference (or mass-to-charge ratio) of the total mass of the multi-dimensional signal may be used.

For use in the analysis of a given indicator level, it is beneficial that the multidimensional signal used and the multiple sets of multidimensional signals have associated or closely spaced characteristics. For example, multi-dimensional signals with different mass-to-charge ratios (generating mass patterns) and multiple sets of multi-dimensional signals can have relatively small mass-to-charge ratio differences. This allows the multidimensional signal (and / or protein or other analyte to which the multidimensional signal is added) to be accurately separated from another molecule based on properties (eg, mass to charge ratio), and from each other and from another Dimensional signals and patterns (eg, mass patterns) can be generated. For this reason, a predetermined pattern can be identified more easily.
Common of multidimension signals (eg, reporter signal or indicator signal), multidimension signal / analyte conjugate, fragment conjugate, multidimension signal fusion, multidimension signal fusion fragment, or multidimension signal peptide to form a pattern Preferably, the characteristic, or characteristic of the multidimensional signal (eg, reporter signal or indicator signal) is not an affinity tag. Nevertheless, even in such cases, multidimensional signals, multidimensional signal / analyte conjugates, fragment conjugates, multidimensional signal fusions, multidimensional signal fusion fragments, or multidimensions that have otherwise common characteristics The signal peptide can include an affinity tag and-a multidimensional signal, multidimensional signal / analyte conjugate, fragment conjugate, multidimensional signal fusion, multidimensional signal fusion fragment, or multidimensional signal peptide that shares common properties As long as there is another common property that can be used to separate the molecule from molecules lacking other common properties, or another that can be used to generate the pattern (which may be used, depending on the embodiment of the disclosed method) As long as these properties are present-in fact, they can share the same affinity tag. With this in mind, chromatographic or other separation techniques are based on common characteristics, multidimensional signals, multidimensional signal / analyte conjugates, fragment conjugates, multidimensional signal fusions, multidimensional signal fusion fragments, or multidimensional signal peptides. The affinity is preferably based on the general physical properties of the reporter signal rather than the presence of features or sites such as affinity tags, for example. As used herein, a common property is a set of components (eg, multidimension signal, multidimension signal / analyte conjugate, fragment conjugate, multidimension signal fusion, multidimension signal fusion fragment, or Multidimensional signal peptide). That is, the components have “common” characteristics. It should be understood that multidimension signals, multidimension signal / analyte conjugates, fragment conjugates, multidimension signal fusions, multidimension signal fusion fragments, or multidimension signal peptides within a set can have a number of common properties. It is. However, as used herein, common properties of the referenced multidimensional signal, multidimensional signal / analyte conjugate, fragment conjugate, multidimensional signal fusion, multidimensional signal fusion fragment, or multidimensional signal peptide Distinguish between multidimension signals, multidimension signal / analyte conjugates, fragment conjugates, multidimension signal fusions, multidimension signal fusion fragments, or multidimension signal peptides that share common properties from molecules lacking common properties and / or Only the common properties used in the disclosed method to separate. Further, as used herein, the characteristics of a multidimensional signal (eg, reporter signal and indicator signal) used to generate a pattern (“pattern generation characteristics”) can be used to generate a pattern. Only the properties used in the disclosed method.
The predetermined pattern can include any feature, characteristic, property, etc. of the multidimensional signal. A pattern generally includes differences in features, characteristics, properties, and the like. In particular, the patterns can include differences in, for example, specific, repeatable, unique, predicted, or consistent features, characteristics, characteristics, and the like. For example, the pattern may be a specific mass to charge ratio difference between two or more multidimensional signals. In general, a pattern includes two or more different uniqueness or values such as features, attributes, characteristics, and the like. That is, the pattern generally includes uniqueness or differences between values, features, characteristics, properties, etc. of various multidimensional signals.

  The predetermined pattern of features, characteristics, properties, etc. of the multidimensional signal can be formed from a beneficial or desired combination of uniqueness or values, such as features, characteristics, properties. For example, two, two or more, three, three, four, four, five, five, five, six, six or more, seven, seven or more of these numbers of uniqueness or values such as features, characteristics, properties, etc. 8, 8 or more, 9, 9 or more, 10, 10 or more, 11, 11 or more, 1212, 12 or more, 13, 13 or more, 14, 14 or more, 15, 15 or more, 16, 16 or more, 17, 17 or more, 18, 18 or more, 19, 19 or more, 20, 20 or more, 21, 21 or more, 22, 22 or more, 23, 23 or more, 24, 24 or more, 25, 25 or more, 26, 26 or more, 27, 27 or more, 28, 28 or more, 29, 29 or more, 30, 30 or more, 35, 35 or more, 40, 40 or more, 45, 45 or more, 50, 50 or more, 55, 55 or more, 60, 60 or more, 65, 65 or more, 70, 70 or more, 75, 75 or more, 80, 80 or more, 85, 5 above, 90, 90 or more, 95, 95 or more, 100, 100 or more, or any combination, can be used as the predetermined pattern.

  A variety of different properties separate patterns from multidimensional signals, patterns for indicator-level analysis, or molecules that lack other common properties (eg, reporter signals or indicator signals), or multidimensional It can be used as a physical property used to generate a pattern that separates signal / analyte conjugates, fragment conjugates, multidimensional signal fusions, multidimensional signal fusion fragments and / or multidimensional signal peptides. For example, non-limiting physical properties that are useful as a pattern of common properties include mass, charge, isoelectric point, hydrophobicity, chromatographic properties, density, and the like. In certain embodiments, depending on the physical characteristics used to generate the pattern, or multidimensional signal / analyte conjugate, fragment conjugate, multidimensional signal fusion, multidimensional signal fusion fragment or multidimensional signal peptide in the set A physical property that is shared (and used to distinguish or separate multidimensional signal / analyte conjugate fragment conjugates, multidimensional signal fusions, multidimensional signal fusion fragments or multidimensional signal peptides) is simply a feature or Multidimensional signal, multidimensional signal / analyte conjugate, fragment conjugate, multidimensional signal fusion, multidimensional signal fusion fragment and / or multidimensional signal peptide, not the presence of a site (eg, an affinity tag such as biotin) Characteristics (eg, total mass, total charge, isoelectric point, total hydrophobicity, etc.). The above properties are referred to herein as “overall” properties (thus multidimensional signal / analyte conjugates, fragment conjugates, multidimensional signal fusions, multidimensional signal fusion fragments or multidimensional signal peptides within a set , Referred to as sharing a “common overall characteristic”). Multidimension signals (eg, reporter signals or indicator signals), multidimension signal / analyte conjugates, fragment conjugates, multidimension signal fusions, multidimension signal fusion fragments and / or multidimension signal peptides are characterized by features such as affinity tags and It should be understood that the features and sites can contribute to overall properties (eg, by contributing to mass). However, the limited and isolated features and sites described above generally do not serve as the sole basis for overall properties.

  Multiple sets of multidimensional signals (eg, reporter signals and indicator signals) can have any number of multidimensional signals. For example, multiple sets of multidimensional signals are one, two or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more. , 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, or 500 or more different multidimensional signals. A specific number of multidimensional signals and a specific end point of a range of multidimensional signals are listed, but each one of a specific number of multidimensional signals and a specific end point of a range of multidimensional signals One is not explicitly listed, and is intended to specifically describe each specific number of multidimensional signals and each specific end point of a multi-field multidimensional signal range. .

Multiple sets of multidimensional signals can be composed of multidimensional signals consisting of chains or polymers. The multidimensional signal of the set may be a homoset meaning that the set consists of one type of multidimensional signal, or that the multidimensional signal consists of a homochain or a homopolymer. A set of multidimensional signals may be a heteroset, meaning that the set consists of different multidimensional signals, or that the multidimensional signal consists of various types of chains or polymers. A special kind of heteroset is a set consisting of different homochains or homopolymers, eg one peptide chain and one nucleic acid chain. Another special type of heteroset is a set in which the chain itself is a heterochain or heteropolymer. Yet another type of heteroset is a set consisting of both heterochains / heteropolymers and homochains / homopolymers.
The disclosed multidimensional signals can be associated with the analyte or protein, incorporated into the analyte or protein, or otherwise bound. If there is no substantial physical association between the multidimensional signal and the analyte, or between the multidimensional signal and the protein, or by itself, no analyte or protein is present, the multidimensional signal is the analyte or protein (eg, multidimensional It can also be bound within the signal and analyte or protein mixture).

  When a multidimensional signal is no longer associated with an analyte or protein, multiple sets of multidimensional signals have one or more characteristics that cause two or more multidimensional signals in the set to generate a pattern in indicator level analysis Sometimes available. Further, if the reporter signal is no longer associated with the analyte, the multiple sets of reporter signals can distinguish between reporter signals in which two or more reporter signals in the set have a common property from molecules lacking other common properties and / or It can be used when it has one or more common properties that can be separated. Detection of a multidimensional signal indicates the presence of the corresponding analyte or protein.

  Multidimensional signals are preferably detected using mass spectrometry that allows sensitive discrimination between molecules based on their mass-to-charge ratio. The disclosed multidimensional signals can be used as general labels in myriad labeling and / or detection techniques.

  Some types of multidimensional signals (eg, reporter signals or indicator signals) can include one or more affinity tags. The affinity tag allows detection, separation, sorting, or other manipulation of labeled proteins, labeled analytes, multidimensional signals, multidimensional signal fragments, or multidimensional signal fusions based on affinity tags. With respect to the indicator signal, the affinity tag is separated from and in addition to the properties (not the basis of the properties) of the set of indicator signals used to generate the pattern. Rather, the affinity tag serves the different purpose of making the sample operable prior to or as part of the disclosed method, rather than a means of separating the indicator signal based on pattern generation characteristics. With respect to the reporter signal, the affinity tag is separated from and in addition to the common properties of the set of reporter signals that allow separation of the reporter signal from another molecule (rather than the basis for the common properties). . Rather, the affinity tag serves the different purpose of making the sample operable prior to or as part of the disclosed method, rather than a means of separating reporter signals based on common properties.

  A multidimensional signal (eg, a reporter signal or indicator signal) can have zero, one, or more than one affinity tag. If the multidimensional signal has multiple affinity tags, the tags on a given multidimensional signal may all be the same or a combination of different affinity tags. In accordance with the principles described above and elsewhere herein, affinity tags are used to differentially change mass and / or charge on the indicator signal, and mass and / or charge on the reporter tag. Can also be used to be distributed differentially. Affinity tags can be used with multidimensional signals in a manner similar to the use of affinity tags as described in PCT application WO 00/11208.

  Special peptides such as peptide-DNA conjugates (Olejnik et al., Nucleic Acids Res., 27 (23): 4626-31 (1999)), synthesis of PNA-DNA constructs, and the photolabile universal nucleotides of WO 00/04036 Nucleotides can be used as indicator signals in the disclosed methods. Useful photolabile linkages are described by Marriot and Ottl, “Synthesis and application of heterobifunctional photolabile cross-linking reagents”, Methods Enzymol. 291: 155-75 (1998).

Since the multidimensional signal can be precisely controlled and released from the analyte or protein (or other mediator molecule) to which the multidimensional signal is added, photodissociative binding and ligation occurs in the multidimensional signal (and Useful for use with dimensional signals). Various photolabile bonds and linkages are known and can be adapted for use in and with indicator signals. In recent years, photolabile amino acids have become commercially available. For example, Fmoc-protected photolabile slightly modified phenylalanine (Fmoc-D, L-β Phe (2-NO ₂ )) is available (catalog number 0011-F; Innovachem, Tucson, AZ). By introducing a nitro group into the phenylalanine ring, amino acids are fragmented under irradiation with ultraviolet light (at a wavelength of about 350 nm). A nitrogen laser emits light at about 337 nm and can be used for fragmentation. The wavelength used does not cause significant damage to the rest of the peptide.

  Fmoc synthesis is a common technique for peptide synthesis, and this technique can be used to incorporate Fmoc-derived photolabile amino acids into peptides. Photolabile amino acids can be used within and with any multidimensional signal, but are particularly useful with peptide multidimensional signals (eg, peptide reporter signals and peptide indicator signals).

  The use of photolabile bonds and linkages in and with multidimensional signals can be illustrated by the following examples. The material on a blank plastic substrate (eg, a compact disc (CD)) can be measured directly from its surface using a MALDI source ion trap. For example, a thin portion of a tissue sample can be flash frozen and applied to a CD surface. A multidimensional signal molecule (eg, an antibody to which a multidimensional signal has been added via a photolabile linkage) can be applied to the tissue surface. By recognizing specific components within the tissue, a portion of the antibody / multidimensional signal conjugate can be associated (excess conjugate is removed in a subsequent wash step). The multidimensional signal can then be released from the antibody by irradiation with ultraviolet light and detected directly using a MALDI ion trap instrument.

For example, a peptide of the sequence CF ^* XXXXXXDPXXXXXXR (SEQ ID NO: 9) (including the reporter signal) can be added to the antibody using the disulfide bond linkage method. When irradiated with a UV source of a MALDI laser, the peptide is cleaved with a modified phenylalanine, F ^* , releasing the XXXXXXDPXXXXXX reporter signal (amino acids 3-15 of SEQ ID NO: 9). The reporter signal can then be fragmented with DP binding and charge fragments detected as described elsewhere herein. In another example, a peptide of the sequence CF ^* XXXXXXXXXXXXR (SEQ ID NO: 12) (including indicator signal) can be added to the antibody using a disulfide bond linkage method. Irradiation with a MALDI laser UV source cleaves the peptide with a modified phenylalanine, F ^* and releases the XXXXXXXXXXXXR indicator signal (amino acids 3-15 of SEQ ID NO: 12).

  Another example of the use of a photolabile linkage with a multidimensional signal includes DNA-peptide chimeras used as multidimensional signal molecules. The multidimensional signal molecule is effective as a probe for detecting a specific nucleic acid sequence. In a DNA-peptide chimera (or PNA-peptide chimera), the peptide portion may be a multidimensional signal or may contain a multidimensional signal. When the photolabile phenylalanine is arranged, for example, in the vicinity of the junction between the DNA peptide and the multidimensional signal molecule, the multidimensional signal can be released from the multidimensional signal molecule by ultraviolet light. The released multidimension signal can be detected directly or fragmented and detected as described elsewhere herein. As with the antibody-peptide multidimensional signal molecule described above, the DNA-peptide chimera is associated with the released multidimensional signal using a nucleic acid molecule present on the surface of a substrate such as a CD and a UV source of a MALDI laser. Can do.

  To achieve various effects, multiple photolabile bonds and / or linkages can be within or together with the same multi-dimensional signal or multi-dimensional signal conjugate (eg, multi-dimensional signal molecule or multi-dimensional signal fusion) Can be used. For example, different photolabile linkages cleaved by different wavelengths of light can be used in different parts of a multidimensional signal or multidimensional signal conjugate that are cleaved at different stages of the method. Thanks to the various fragmentation wavelengths, for example, sequential processing can be performed which allows a combination of release and fragmentation methods.

As an example, a peptide containing two photolabile amino acids, Z (infrared cleavage wavelength) and F ^* (photodissociable phenylalanine, ultraviolet cleavage wavelength) uses a known chemical reaction at the amino terminus. It can consist of the format XZXXXXXXXF ^* XXXXXXXR that can be attached to an analyte or other molecule. The result is a reporter signal / analyte conjugate (or reporter molecule) or an indicator signal / analyte conjugate (or indicator molecule). The multidimensional signal can be released from the conjugate by irradiating the conjugate with the appropriate wavelength of light (infrared in this example), thereby breaking the bond at Z. Once the parent ion is selected and stored in the ion trap, the multidimensional signal is fragmented by irradiating with the appropriate wavelength of light (in this example, ultraviolet light) and the daughter ion (XXXXXXXR ⁺ ) that can be detected and quantified. create.

  Other labels that can be used as multidimension signals, reporter signals and / or indicator signals are US applications 2004/0018565, 2003/0100018, 2003/0050453, 2004/0023274, 2002/014673, 2003 / 0022225, and US Pat. Nos. 6,312,893, 6,312,904, 6,629,040, and Geysen et al. (Chemistry & Biology 3 (8): 679-688 (1996)), all of which are incorporated herein by reference.

Multidimensional signals can be added, linked, or immobilized to any desired analyte, compound, substrate, or other composition using any suitable technique. As used herein, molecules are linked when they are covalently bonded directly or indirectly. One form of indirect attachment is through a linker molecule. A multidimensional signal can be bound to an analyte, compound, substrate, or other composition by any suitable binding reaction. Many chemicals and techniques for binding compounds are known and can be used to link multidimensional signals to an analyte. For example, conjugation can be performed using thiols, epoxides, thiols for thiols, NHS esters, isothiocyanates, isothiocyanates for amines, amines, and alcohols for carboxylic acids. In another example, peptide multidimensional signals that can be linked via acetylation of primary amines are known (Wetzel et al., Bioconjugate Chem1, 114-122 (1990)).
B. Reporter signal A reporter signal (also called a reporter signal peptide) is a molecule that is preferentially fragmented, degraded, reacted, derivatized, or otherwise modified or altered for detection. Reporter signal is a form of multidimensional signal.
References to derivatives having the properties of multidimensional signals and reporter signals and their derivatives can be considered the same as the reporter signal version of the multidimensional signal (and in this situation the labels can be interchanged). Detection of the modified reporter signal is preferably accomplished by mass spectrometry. The disclosed reporter signals are preferably used in sets where the members of the set have the same mass to charge ratio (m / z). This simplifies sensitive filtering or separation of the reporter signal from another molecule based on the mass to charge ratio. The reporter signal can take any structure that allows for identification of a modified reporter signal that is different from the modification of the reporter signal. The reporter signal is preferably configured such that at least one preferential bond breakage is induced in the molecule. A set of reporter signals having nominally the same molecular mass and an arbitrarily selected internal fragmentation point can be configured such that after fragmentation, each member of the set yields a daughter fragment with a unique correlation. . For convenience, reporter signals that have been fragmented, degraded, reacted, derivatized, or otherwise modified for detection are referred to as fragmented reporter signals. A suitable reporter signal can be fragmented by tandem mass spectrometry.

  Reporter signals are preferably used in sets where all reporter signals in the set have similar physical properties. Similar (or common) properties can distinguish and / or separate reporter signals from other molecules that lack one or more properties. Preferably, the reporter signals within the set have the same mass to charge ratio (m / z). That is, the reporter signal within the set can be isobaric. This allows the reporter signal (and / or the protein or other analyte to which the reporter signal is added) to be accurately separated from another molecule based on the mass to charge ratio. As a result of filtering, the signal-to-noise ratio (S / N) for the system is greatly increased, allowing for more sensitive and accurate detection. The set of reporter signals can have any number of reporter signals.

  Preferred general properties are those of subunit isomers. This property is that a set of at least two reporter signals (usually composed of subunit chains composed of subunits, such as the relationship between a polymer and a unit comprising a polymer) is a subunit isomer. And the set can be referred to as subunit isomers or subunit isomers. Although subunits are discussed elsewhere in this specification, the reporter signal can be composed of any type of chain, such as peptides or nucleic acids or polymers (generally), which are then subunits such as amino acids and phosphos. Each can be composed of a nucleoside and a mer (general). Within each type of subunit, there are usually multiple members that are all of the same type but different. For example, within the subunit type “amino acid” there are many members, eg, ala, tyr, and ser, or other amino acid combinations.

  When a set of reporter signals are subunit isomers or are composed of subunit isomers, it means that each individual in the set is a subunit isomer of all other individual subunits in the set . By isomers is meant that the subunits forming the subunit chain have the same configuration (ie, distribution or array), but the overall connectivity of the subunits forming the chain is different. Thus, for example, the first reporter signal is the chain ala-ser-lys-gln, the second reporter signal is the chain ala-lys-ser-gln, and the third reporter signal is the chain ala-ser-lys-pro. There may be. When a set of reporter signals is generated comprising a first reporter signal and a second reporter signal, the first reporter signal and the second reporter signal have the same configuration, i.e. each has one ala The set is a subunit isomer because it has one ser, one lys, and one gln, but each chain has a different connectivity. However, when a set of reporter signals is generated comprising a first, second, and third reporter signal, the configuration of each strand is not identical and the first and second strands do not have pro. First, the set does not become an isomer because the third strand has no gin.

  Another example is as follows. The first reporter signal may be chain ala-guanosine-lys-adenosine, the second reporter signal may be chain ala-adenosine-lys-guanosine, and the third reporter signal may be chain ala-ser-lys-pro. . When a set of reporter signals is generated comprising a first reporter signal and a second reporter signal, the first reporter signal and the second reporter signal have the same configuration, i.e. each has one ala Having one guanosine, one lys, and one adenosine, but each chain has a different connectivity, so the set is a subunit isomer. However, when a set of reporter signals is generated comprising a first, second, and third reporter signal, the configuration of each strand is not identical and the first and second strands have pro or ser And the third strand does not have guanosine or adenosine, so the set is not an isomer. As this example shows, the set can consist of or contain heterochains and still be considered subunit isomers.

  Reporter signals within a set can be fragmented, degraded, reacted, derivatized, or otherwise modified or altered to distinguish different reporter signals within the set. Preferably, the reporter signal is fragmented to yield fragments of similar charge but different mass. Reporter signals can also be fragmented to produce fragments of different charge and mass. With the above modifications, each reporter signal in the set can be distinguished by different mass to charge ratios of the reporter signal fragments. This is possible because non-fragmented reporter signals within a set can be isobaric, but fragments of different reporter signals are non-isobaric. Thus, a key feature of the disclosed reporter signal is that the reporter signal has similar properties while the modified reporter signal is distinguishable.

Differential mass distribution in reporter signal fragments can be achieved in a variety of ways. For example, different distributions of reporter signals of the same nominal structure (eg, peptides having the same amino acid sequence), deuterium ( ² H), tritium ( ³ H) ¹⁷ O, ¹⁸ O, ¹³ C, or ¹⁴ C The stable isotopes are preferred. All reporter signals in the set have the same number of given heavy isotopes, but their distribution depends on the various reporter signals. Examples of the set of reporter signals are A ^* G ^* SLDPAGSLR, A ^* GSDLDPAG ^* SLR, and AGSLDPA ^* G ^* SLR (SEQ ID NO: 2), where the asterisk is at least one heavy isotope with an amino acid substitution Indicates. After fragmentation with a single charged parent ion and a cleaved DP bond, for one primary charged daughter ion, three distinct primary daughter ions, PAGSLR ⁺ , PAG ^* SLR ⁺ , PA ^* G ^* SLR ⁺ (SEQ ID NO: 2 amino acids 6-11).

Similarly, reporter signals of the same general structure (eg, peptides having the same amino acid sequence) can be modified by methylation, phosphorylation, sulfation, and other modifications or different distributions of substituents, and seleno-methionine for methionine. Can be created with use. All reporter signals in the set have the same number of given modifications, but their distribution depends on the various reporter signals. Examples of the above set of reporter signals are AGS ^* M * LDPAGSMLR, AGS * MLDPAGSM * LR, and AGS ^* MLDPAGS ^* M ^* LR (SEQ ID NO: 3), where S ^* is not serine but phosphoserine, M ^* Represents seleno-methionine, not methionine. After fragmentation with a single charged parent ion and a cleaved DP bond, for one primary charged daughter ion, three distinct primary daughter ions, PAGSMLR ⁺ , PAGSM ^* LR ⁺ , PAGS ^* M ^* LR ⁺ (SEQ ID NO: 3 amino acids 7-13).

Reporter signals of the same nominal composition (eg, composed of the same amino acids) can be generated with different sequence subunit or component reporter signals. All reporter signals in the set have the same number of subunits or components, but their distribution depends on the various reporter signals. Examples of the one set of reporter signals are AGSLADPGSLR (SEQ ID NO: 4), ALSLADPGSGR (SEQ ID NO: 5), and ALSLGDPASGR (SEQ ID NO: 6). After fragmentation with a single charged parent ion and a cleaved DP bond, for one primary charged daughter ion, three distinct primary daughter ions, PGSLR ⁺ (amino acids 7-11 of SEQ ID NO: 4), PGSGR ⁺ (SEQ ID NO: 5 amino acids 7 to 11), and PASG R ⁺ (SEQ ID NO: 6 amino acids 7 to 11).

Reporter signals having the same nominal composition (eg, composed of the same amino acids) can be generated with unstable or cleavable bonds at various positions of the reporter signal. All reporter signals in the set have the same number and order of subunits or components. When unstable or cleaved bonds are present between specific subunits or components, the sequence of the subunits or components in the reporter signal is the same except for the subunits or components that form unstable or cleaved bonds. There may be. Reporter signal peptides used in reporter signal fusion preferably use this form of differential mass distribution. Examples of the one set of reporter signals are AGSLADPSLR (SEQ ID NO: 4), AGSDPLAGSLR (SEQ ID NO: 7), and ADPGSLAGSL (SEQ ID NO: 8). After fragmentation with a single charged parent ion and a cleaved DP bond, for one primary charged daughter ion, three distinct primary daughter ions, PGSLR + (amino acids 7-11 of SEQ ID NO: 4), PLAGSLR ⁺ ( SEQ ID NO: 7 amino acids 5-11), PGSLAGSLR ⁺ (SEQ ID NO: 8 amino acids 3-11).

Each of these modes can be combined with one or more other modes to generate a differential mass distribution of reporter signal fragments. For example, different distributions of heavy isotopes can be used in reporter signals where unstable or cleavable bonds are made at different positions. Different mass distributions can be achieved in other ways. For example, the reporter signal can introduce various modifications at different positions. Examples of effective modifications are methionine, not sulfation, but acetylation, methylation, phosphorylation, seleno-methionine. Similar principles can be used to differentially distribute charge with a reporter signal. Mass and charge differential distributions can be used together in multiple sets of reporter signals.
The reporter signal can also include a combination of a cleavage bond and a labile bond. Thereby, the combination of signals that can be distinguished can be increased, or detection can be simplified. For example, labile bonds can be used to release isobaric fragments and cleaved bonds can be used to decode proteins.

  Selenium substitution can be used to alter the mass of the reporter signal. Selenium can substitute for sulfur in methionine to give the modified amino acid selenomethionine. Selenium is about 47 mass units larger than sulfur. Mass spectrometry can be used to identify peptides or proteins that incorporate selenomethionine and methionine in a specific ratio. Small proteins and peptides with known selenium / sulfur ratios are preferably made by chemical synthesis incorporating selenomethionine and methionine in the desired ratio. Large proteins or peptides can be generated from an E. coli expression system that inserts selenomethionine and methionine in the desired ratio, or other expression systems (Hendrickson et al., “For analysis by multi-wavelength abnormal diffraction (MAD). Selenomethionine protein prepared in: 3D structure direct determination means ”Embo J, 9 (5): 1665-72 (1990), Cowie and Cohen,“ Escherichia coli of an activity-modifying protein containing selenium instead of sulfur Synthesis ", Biochimica et Biophysica Acta, 26: 252-261 (1957), and Oikawa et al.," Metaloselenonein, a selenium analog of metallothionein: synthesis and characterization of complexes with copper ions "Proc Natl Acad Sci USA, 88 (8): 3057-9 (1991).

As described above, the reporter signal can include a photolabile linkage that precisely controls and releases the reporter signal from the analyte or protein (or other mediator molecule) to which the reporter signal is added. Photolabile linkages can also be incorporated into reporter signals and used for reporter signal fragmentation in the disclosed manner. For example, a photolabile amino acid (such as a photolabile phenylalanine) can be incorporated at the desired position of the peptide reporter signal. Reporter signals such as XXXXXXF ^* XXXXR containing photolabile phenylalanine (F ^* ) are photolabile. The reporter signal can then be fragmented using light of the appropriate wavelength and the detected charge fragment. When ionizing a reporter signal (eg, from the surface) for detection, a MALDI laser that does not cause significant photodissociation (eg, Er: YAG at 2.94 μm) can be used for ionization, Lasers (eg, nitrogen at 337 nm) can be used to fragment the reporter signal. In this case, XXXXXXFXXXXXXR ⁺ is photodissociated to produce XXXXXXR +. The second laser can intersect the reporter signal ion packet at any location. The modification of the mass spectrometer vacuum system for this purpose is simple.

The use of a photolabile ligation in the reporter signal is particularly effective when the analyte or protein (or other component) to which the reporter signal is added can be cleaved and fragmented in the collision cell. For example, in a reporter signal fusion, a protein fragment / reporter signal polypeptide can be generated to include a cleavage bond at both the protein fragment portion and the reporter signal portion. One example is XXXXXXXXXXXXDPXXX (XXXXXXXXXDPXXXXXXXXXR) XXXX (SEQ ID NO: 10), the sequence in parentheses indicates that the reporter signal moiety and DP dipeptide contain a cleavage bond, and X is any amino acid. Fragmenting this polypeptide within the collision cell complicates the fragment spectrum by causing fragmentation at one or both of the DP bonds. The use of a photolabile linkage (eg, a photolabile amino acid) in the reporter signal moiety allows for specific photodissociation of the reporter signal under analysis. For example, the similar polypeptide XXXXXXXXXXDPXXX (XXXXXXXF ^* XXXXXXXR) XXXX (SEQ ID NO: 11) allows specific photodissociation of the F ^* position of the reporter signal.

  Reporter signal calibrators are a special form of reporter signal characterized by use in reporter signal calibration. The reporter signal calibrator may be any form of reporter signal, as described above and elsewhere herein, but is a separate molecule that is not physically associated with the analyte or protein being evaluated. Used as. Thus, the reporter signal calibrator need not have (preferably not have) a reactive group for binding to the analyte or protein, and can be a specific binding molecule or another described herein as associated with the reporter signal. Need not be associated (preferably not associated) with any molecule or component.

  The reporter signal calibrator preferably shares one or more common characteristics with one or more analytes. Reporter signal calibrators and analytes that share one or more common characteristics are referred to as a reporter signal calibrator / analyte set. If only one analyte and one reporter signal calibrator share common characteristics, they can also be referred to as a reporter signal calibrator / analyte pair. The reporter signal calibrator and the analyte in the reporter signal calibrator / analyte set are said to match. The common property allows the reporter signal calibrator and its matching analyte to be distinguished and / or separated from another molecule lacking one or more properties. Preferably, the reporter signal calibrator and the analyte in the set have the same mass to charge ratio (m / z). That is, the matching reporter signal calibrator and analyte in the set may be isobaric. This allows the reporter signal calibrator and the analyte to be accurately separated from other molecules based on the mass to charge ratio. The reporter signal calibrator can be fragmented, degraded, reacted, derivatized, or otherwise modified or altered to distinguish the altered reporter signal calibrator from the matching analyte. Samples can also be fragmented. Reporter signal calibrators can be fragmented to produce fragments of similar charge but different mass, or can be fragmented to produce fragments of different charge and mass. With the above changes, reporter signal calibrators (and other analytes and / or reporter signal calibrators, if any, that are members of the same set) are distinguished from matching analytes by different mass to charge ratios of reporter signal calibrator fragments. Can do. This is possible because the non-fragmented reporter signal calibrator and analyte in the set are isobaric, but the reporter signal calibrator fragments are not isobaric. Thus, a key feature of the disclosed reporter signal calibrator is that it has similar characteristics to the analyte that the reporter signal calibrator matches and is distinguishable from the analyte that the modified reporter signal calibrator matches.

  Suitable analytes for use with the reporter signal calibrator are proteins, peptides, and / or protein fragments (collectively referred to as proteins for convenience). Reporter signal calibrators and proteins that share one or more common properties are referred to as a reporter signal calibrator / protein set. When only one protein and one reporter signal calibrator share a common property, they are also referred to as a reporter signal calibrator / protein pair. Reporter signal calibrators and proteins in the reporter signal calibrator / analyte set are said to match.

  As described elsewhere herein, the reporter signal calibrator can use the analyte in the sample as a basis for assessing the presence and amount. For this purpose, a reporter signal calibrator designed for each analyte to be evaluated can be mixed with the sample to be analyzed. Next, reporter signal calibrators that match the analyte are processed together to detect both the analyte and the reporter signal calibrator (preferably in a modified format). The amount of reporter signal calibrator detected or altered reporter signal calibrator can compare the amount of either detected or altered analyte (because the amount of added reporter signal calibrator is known) Provide criteria for what can be done. Thereby, the amount of the specimen present in the sample can be accurately measured.

i-PROT labels can be used as multidimensional signals and reporter signals in the disclosed compositions and methods. The i-PROT system and label are referred to as reporter signals and are described in US Application No. 2003/0194717, US Application No. 2004/0220412, US Application No. 2003/0124595, and US Pat. No. 6,824,981. All of which are hereby incorporated by reference for the description of reporter signals and the use of reporter signals for labeling and detection. In the i-PROT system, the reporter signal can be added to an analyte such as a protein by any method.
In i-PROT systems, the reporter signal is preferably fragmented to produce fragments of similar charge but different mass. This allows each labeled analyte (and / or each reporter signal) in the set to be distinguished by different mass to charge ratios of the reporter signal fragments. This is possible because the non-fragmented reporter signals in the set are isobaric, but fragments of different reporter signals are not isobaric.

  In i-PROT systems, reporter signals can be used in sets where all reporter signals in the set have similar properties (eg, similar mass to charge ratios). Similar properties allow the reporter signal to be distinguished and / or separated from another molecule lacking one or more properties. Preferably, the reporter signals within the set have the same mass to charge ratio (m / z). That is, the reporter signal within the set is isobaric.

  iTRAQ labels can be used as multidimensional signals and reporter signals in the disclosed compositions and methods. The iTRAQ system and label are described in US Application No. 2004/0220412 and PCT Application No. WO 2004/070352, both of which are described herein for the description of iTRAQ label and the use of iTRAQ label for labeling and detection. Include in the book. iTRAQ is a labeling system that uses multiplexed sets of reagents for quantitative protein analysis that place isobaric mass labels on the N-terminus and lysine side chains of peptides in the digestion mixture. All derivatized peptides are isobaric and chromatographically indistinguishable, but produce a signature or reporter ion after CID that can be used to identify and weigh individual members of multiple sets. Reagents are differentially isotopically labeled. Thus, iTRAQ label is a kind of reporter signal. iTRAQ label is an amino-specific stable isotope reagent that allows relative and absolute quantification from MS / MS spectra by allowing simultaneous labeling of all peptides in up to four different biological samples . In the iTRAQ system, the reporter is a 5, 6 or 7 membered heterocycle with a ring nitrogen atom where the analyte is N-alkylated at the substituted or unsubstituted acetic acid moiety attached through the carbonyl carbon of the N-alkylacetic acid moiety. And each different label comprises one or more heavy atom isotopes. The heterocycle may be substituted or unsubstituted. Heterocycles are aliphatic or aromatic. Possible substituents for the heterocyclic moiety are alkyl groups, alkoxy groups, and allyl groups. Substituents can comprise protected or unprotected groups such as amine groups, hydroxyl groups, or thiol groups suitable for binding the supporting analyte. Heterocycles can have one or more additional heteroatoms such as nitrogen, oxygen, or sulfur atoms.

  The components of an example of iTRAQ labeled multiple derivatization chemistry are shown in FIGS. Ross et al. , MCP Paper in Press, Manusscript M400129-MCP200 (September 28, 2004), the six protein reductive alkylation digest mixture is divided into four identical aliquots. Ross et al. Is incorporated herein by reference for the description of iTRAQ labels and the use of iTRAQ labels for labeling and detection. Each is then labeled with one of four isotope-labeled tags and derivatized into digests that are bound to the mixture in various proportions. Multiple isobaric tags produced abundant MS / MS signature ions at 114.1, 115.1, 116.1, 117.1 m / z, the relative area of these peaks corresponding to the proportion of labeled peptide.

  The mass shift imposed by the isotopic enrichment of each signature ion in this example of iTRAQ balances the isotopic enrichment with the carbonyl component of the derivative so that the total mass of each of the four tags is the same. Thus, a given peptide labeled with each of the four tags has the same integer mass, providing a sensitivity enhancement over mass difference labeling. For isobaric peptides, the MS ionic current at a given peptide mass is the sum of the ionic currents from all samples in the mixture, so the spectrum of the spectrum by combining the MS precursor signal and combining two or more samples No complication occurs (FIGS. 6 and 7). Since all peptide main chain fragment ions are isobaric, isobaric and sensitivity enhancement is carried over to the MS / MS spectrum (FIG. 7).

FIG. 6 shows a structural diagram of the iTRAQ multiple isobaric labeling chemical reaction. FIG. 6A shows a complete molecule consisting of a reporter group (based on N-methylpiperazine), a mass balance group (carbonyl) and a peptide reactive group (NHS ester). The reporter's total mass and molecular balance components are kept constant using different isotopic enrichments of ¹³ C and ¹⁸⁰ atoms, avoiding the problems associated with chromatographic separations seen in enrichment involving deuterium substitution. The mass of the reporter group ranges from 114.1 to 117.1 m / z and the mass of the balance group ranges from 28 to 31 daltons so that the combined mass of the four reagents is constant (145.1 daltons). FIG. 6B shows the structure when the tag reacts with the peptide to form an amide linkage to the peptide amine (N-terminal or epsilon amino group of lysine). Upon undergoing collision-induced dissociation (CID), these amide-linked fragments break in the same way as backbone peptide bonds. However, after fragmentation of the tag amide bond, the balance (carbonyl) site is lost (neutral loss) and the charge is retained by the reporter group fragment. FIG. 6C shows isotope tagging used to arrive at four isobaric combinations with four different reporter group masses (left). A mixture of four identical peptides, each labeled with one member of the multiplex set, appears as a single undivided precursor ion in the MS (identical m / z; middle). After collision-induced dissociation (CID), the four reporter group ions appear as separate masses (114-117 daltons; right). All other sequence-information fragment ions (b-, y-, etc.) remain isobaric and their individual ionic current signals (signal intensity) are additive. This is also the case for peptides that contain an internal lysine residue due to incomplete cleavage of trypsin with trypsin peptides that are labeled at both the N-terminus and the lysine side chain. Thus, the relative concentration of the peptide is estimated from the relative intensity of the corresponding reporter ion. Weighing is performed at the MS / MS stage, not the MS.

In FIG. 7, the peptide TPHPALTAK from the protein digestion mixture made by labeling four separate digests with each of the four isobaric reagents and combining the reaction mixture in a 1: 1: 1: 1 ratio. An example of an MS / MS spectrum is shown. The isotope distribution of the precursor ([M + H] +, 1352.84 m / z) is shown in i). The components within the spectrum frame shown in the center are shown at the bottom. ii) shows the low mass region indicating the signature ion used for quantification, iii) shows the isotope distribution of the b ₆ fragment, and iv) shows the isotope distribution of the Y ₇ fragment. Peptides are labeled with isobaric tags at both the N-terminal and C-terminal lysine side chains. Thus, all of the precursor ions and internal fragment ions (eg, type b- and type y-) contain all four members in the tag set, but remain isobaric. The example shown is a spectrum obtained from a single charge [M + H] + peptide using a 4700 MALDITF-TOF analyzer, but the same is true for any multiple charge peptide analyzed on an ESI-source mass spectrometer.

TMT labels can be used as multidimensional signals and reporter signals in the disclosed compositions and methods. The TMT system is described in US application 2003/0194717, which is incorporated herein by reference for the description of TMT labels and the use of TMT labels for labeling and detection. TMT, or tandem mass tag, is a chemical mass tag that has an individual fragmentation pattern in tandem mass spectrometry. TMT labels can be used as multidimensional signals and reporter signals in the disclosed compositions and methods. Each TMT is in series with a mass reporter group (M) or (M ′), a pro-fragmented linker group (F), a mass normalization group (N) or (N ′) and an amine reactive group (MF— N- (R) first tag; and M′-FN ′-(R) second tag). All members of the series have the same total mass and physicochemical properties ensuring co-elution during chromatography and mass spectrometry. When the labeled peptide enters the tandem MS ion beam, the TMT 'pro-fragmenting element is released, producing a unique mass-to-charge signal.
C. Indicator signal An indicator signal is a molecule having at least one characteristic that can distinguish and / or separate an indicator signal from another multidimensional signal. In general, the indicator signal need only be distinguishable and / or separable from other multidimensional signals present in the same level of analysis. Indicator signals can be used in the set. Thus, for example, a set of indicator signals that differ in several properties or characteristics can be used to label different samples and / or analytes. Depending on the format of the indicator signal, the feature may be a feature of a reporter signal and / or another multidimensional signal used in the same assay or assay system so that a recognizable pattern results during the analysis of the multidimensional signal. It is selected to be compatible with. For example, the indicator signal or multiple sets of indicator signals have a mass (or mass to charge ratio) that is different from the mass of the reporter signal (or mass to charge ratio), and multiple sets of reporter signals are used in the same assay, A unique pattern of mass (or mass to charge ratio) in the analysis can be generated.

  The disclosed indicator signals are preferably used in sets where the members of the set have different mass to charge ratios (m / z). In such a format, the indicator signal preferably has a different mass to charge ratio than another multidimensional signal, such as a reporter signal used in the same assay. This makes it easy to distinguish indicator signals from each other and from other multidimensional signals with high sensitivity based on the mass to charge ratio. The indicator signal can take any structure that can generate a pattern with another multidimensional signal in the analysis of the disclosed method.

  Indicator signals are preferably used in sets where all indicator signals in the set have different physical characteristics. Different (or characteristic) characteristics can distinguish and / or separate the indicator signal from another multidimensional signal that differs in one or more characteristics. Preferably, the indicator signals in the set have different mass to charge ratios (m / z). That is, the indicator signal in the set is non-isobaric. For this reason, the indicator signal (and / or the protein or another analyte to which the indicator signal is added) is accurately separated from another molecule based on the mass-to-charge ratio, producing each other and another multidimensional signal and mass pattern. be able to. The multiple sets of indicator signals can have any number of indicator signals.

  The indicator signals in the set may be subunit isomers, but are preferably not. However, the indicator signal can have portions that are subunit isomers relative to other set member portions and portions that are not subunit isomers relative to other set member portions. The non-subunit isomeric portion of the indicator signal can play a role on the basis of the difference in properties between the members of the set. Thus, for example, the first indicator signal is the chain trp-ala-ser-lys-gln, the second indicator signal is the chain pro-ala-lys-ser-gln, and the third indicator signal is the chain leu-ser- It may be ala-lys-pro. The first indicator signal and the second indicator signal respectively contain a moiety that is a subunit isomer (ala-ser-lys-gln or ala-lys-ser-gln) and a moiety that is not a subunit isomer (trp or leu). Have. The third indicator signal does not share this subunit isomer moiety. However, all three indicator signals have subunit isomer moieties (ala-ser-lys, ala-lys-ser or ser-ala-lys).

  Selenium substitution can be used to change the mass of the indicator signal. Selenium can substitute for sulfur in methionine to give the modified amino acid selenomethionine. Selenium is about 47 mass units larger than sulfur. Mass spectrometry can be used to identify peptides or proteins that incorporate selenomethionine and methionine in a specific ratio. Small proteins and peptides with known selenium / sulfur ratios are preferably made by chemical synthesis incorporating selenomethionine and methionine in the desired ratio. Large proteins or peptides can be generated from an E. coli expression system that inserts selenomethionine and methionine in the desired ratio, or other expression systems (Hendrickson et al., “For analysis by multi-wavelength abnormal diffraction (MAD). Selenomethionine protein prepared in: 3D structure direct determination means ”Embo J, 9 (5): 1665-72 (1990), Cowie and Cohen,“ Escherichia coli of an activity-modifying protein containing selenium instead of sulfur Synthesis ", Biochimica et Biophysica Acta, 26: 252-261 (1957), and Oikawa et al.," Metaloselenonein, a selenium analog of metallothionein: synthesis and characterization of complexes with copper ions "Proc Natl Acad Sci USA, 88 (8): 3057-9 (1991)).

  An indicator signal calibrator is a special type of indicator signal characterized by its use in reporter signal calibration. The indicator signal calibrator may be any form of indicator signal as described above and elsewhere herein, but as a separate molecule that is not physically associated with the analyte or protein being evaluated. used. Thus, the indicator signal calibrator need not have (preferably not have) a reactive group for binding to the analyte or protein, and may be a specific binding molecule or another described herein as being associated with the indicator signal. Need not be associated (preferably not associated) with any molecule or component. The indicator signal calibrator generates a predetermined pattern with the reporter signal calibrator when used together. For reporter signal calibration, the reporter signal calibrator preferably shares one or more common properties with one or more analytes, and the indicator signal calibrator preferably does not share. Rather, the indicator signal calibrator serves to generate a pattern with the reporter signal calibrator.

ICAT labels can be used as multidimensional signals and indicator signals in the disclosed compositions and methods. ICAT systems and reagents (labels) are described in PCT application WO 00/011208, examples using the ICAT system can be found in PCT application WO 02/090929 and US application 2002/0192720, each application being an ICAT label Description and the use of ICAT labels for labeling and detection are incorporated herein by reference. The ICAT label is designed to affinity isolate and quantitate through the use of stable isotope relative concentrations of cysteine-containing tryptic peptides obtained from digests of control versus experimental samples. In certain embodiments, the ICAT reagent has a thiol-specific reactive group comprising adjacent to an alkyl linker comprising either a 9 [ ¹² C] or 9 [ ¹³ C] atom, resulting in the same tryptic peptide of There is a 9 Dalton mass difference between the control version and the corresponding experimental version. The alkyl linker within the ICAT reagent is connected to a (cleavable) biotin group that allows rapid affinity isolation of cysteine-containing tryptic peptides.

Mass defect tags can be used as multidimensional signals and indicator signals in the disclosed compositions and methods. Mass defect tags and their use are described in US Application No. 2002/0172961 and Hall et al. , J .; “Mass Spectrometry” 38: 809-816 (2003), both incorporated herein by reference for the description of mass defect tags and the use of mass defect tags for labeling and detection. A mass defect tag uses an element that includes a larger mass defect that results in a mass-analyzing ionic species having a mass that falls between the masses of ionic species having an integer or nearly integer mass difference. Thus, a mass spectrometry peak having such a non-integer mass is identified as a labeling species and can be distinguished from other peaks. As with other mass labels, the unique mass of a molecule labeled with a mass defect tag can contribute to a predetermined pattern used in indicator level analysis.
Other labels that can be used as multidimensional signals and / or indicator signals are US applications 2004/0018565, 2003/0100018, 2003/0050453, 2004/0023274, 2002/014673, 2003/0022225, And US Pat. Nos. 6,312,893, 6,312,904, 6,629,040, and Geysen et al. (Chemistry & Biology 3 (8): 679-688 (1996)), all of which are incorporated by reference.
D. Analytes and Proteins The disclosed methods generally utilize analytes and proteins as objects for detection, measurement and / or analysis. An analyte is a molecule or part of a molecule that is detected, measured, or otherwise analyzed. A “protein” is a type of analyte and, according to the present invention, includes proteins, peptides, and fragments of proteins or peptides. The analyte or protein need not be physically separate molecules, but may be part of a larger molecule. Analytes include biological molecules, organic molecules, chemicals, constituents, and other molecules or structures to which the disclosed methods can be applied. It should be understood that the different types of disclosed methods are more suitable for some types of analytes than other types of methods. The specimen is also called a target molecule.

A preferred analyte is a biological molecule. Biological molecules include, but are not limited to, proteins, peptides, enzymes, amino acid modifications, protein domains, protein motifs, nucleic acid molecules, nucleic acid sequences, DNA, RNA, mRNA, cDNA, metabolites, carbohydrates, and nucleic acid motifs. . As used herein, “biological molecule” and “biomolecule” refer to a part of a molecular or multimolecular assembly or composition made from a molecule or biological tissue. It relates to a part of the molecular or multimolecular assembly or composition of the material. Biomolecules may be fully artificial molecules related to molecules made from biological tissue.
E. Samples Any sample from any source can be used in the disclosed manner. In general, an analyte sample should be a sample that contains or can contain an analyte. In general, a protein sample should be a sample that contains or can contain protein molecules. Examples of suitable analyte and protein samples include cell samples, tissue samples, cell extracts, components or fragments purified from another sample, environmental samples, biofilm samples, culture samples, tissue samples, body fluids, and cytology It is a sample. Many other sample sources are known or have been developed and can be used with the disclosed methods. Preferred protein samples for use in the disclosed methods are cell and tissue samples. Protein samples can be complex, simple, or in between. For example, a protein sample can include a complex mixture of proteins (eg, a tissue sample), and the protein sample can be a highly purified protein formulation or a single type of protein. Similarly, an analyte sample can include a complex mixture of biological molecules (eg, a tissue sample), and the analyte sample can be a highly purified protein preparation or a single type of molecule.
F. Multidimensional molecule A multidimensional molecule (or multidimensional signal molecule) is a molecule that binds a multidimensional signal to a specific binding molecule or decoding tag. Preferably, the multidimensional signal and the specific binding molecule or decoding tag are covalently linked or linked together. As used herein, molecules are linked when directly or indirectly covalently bound. One form of indirect attachment is through a linker molecule. A multidimensional signal can be linked to a specific binding molecule or decoding tag by several established binding reactions. For example, Hendrickson et al. Nucleic Acids Res. , 23 (3): 522-529 (1995) describe suitable methods for binding oligonucleotides and antibodies. A reporter molecule is a molecule that binds a reporter signal to a specific binding molecule or decoding tag. An indicator molecule is a molecule that binds an indicator signal to a specific binding molecule or decoding tag. Reporter molecules and indicator molecules are in the form of multidimensional molecules.

One type of reporter molecule has a peptide nucleic acid as a decoding tag and a multidimensional signal peptide as a multidimensional signal. A peptide nucleic acid can associate a multidimensional signal peptide with an encoding tag, eg, by associating it with an oligonucleotide encoding tag. As described elsewhere herein, encoded tags can be used for labeled analytes and other molecules.
As used herein, a molecule is said to be connected to another molecule when the loop of one molecule (or from) passes through the loop of (or from) another molecule. The two molecules are not covalently linked when they are joined. The tether can be visualized by analogy with a closed loop string passing through the hole in the mug handle. In general, tethers can be designed so that one or both of the molecules are free to rotate around the loop.
G. Specific binding molecule A specific binding molecule is a molecule that interacts specifically with a specific molecule or site. A molecule or moiety that specifically interacts with a specific binding molecule is referred to herein as an analyte. A preferred specimen is a specimen. The term analyte is understood to refer to both individual molecules that interact specifically with a specific binding molecule and parts of the molecule, such as protein epitopes. Antibodies of receptor / ligand pairs or synthetic polyamides (Dervan and Burli, “Sequence with Polyamide—Specific DNA Recognition”, Curr Opin Chem Biol, 3 (6): 688-93 (1999); Wemmer and Dervan, “DNA , "Target Minor Groove of", Curr Opin Struct Biol, 7 (3): 355-61 (1997)), nucleic acid probes, and other molecules with specific binding affinities are effective binding as an affinity part of a multidimensional molecule. It is an example of a molecule.

  A specific binding molecule that specifically interacts with a particular analyte is said to be specific to that analyte. For example, if a specific binding molecule is an antibody associated with a specific antigen, the specific binding molecule is said to be specific for that antigen. An antigen is a specimen. Multidimensional molecules including specific binding molecules can also be said to be specific to a particular analyte. The specific binding molecule is preferably an antibody, ligand, binding protein, receptor protein, hapten, aptamer, carbohydrates, synthetic polyamides, peptide nucleic acids, or oligonucleotides. A preferred binding protein is a DNA binding protein. Preferred DNA binding proteins are zinc finger motifs, leucine zipper motifs, helix-turn-helix motifs. These motifs can be combined within the same specific binding molecule.

  Antibodies useful as affinity portions of multidimensional molecules are either commercially available or can be made using well-established methods. For example, Johnston and Thorpe, “Practical Immunochemistry” (Blackwell Scientific Publications, Oxford, England, 1987), pages 30-85, provide general methods effective for generating both polyclonal and monoclonal antibodies. Is described. The entire book describes many general techniques and principles for the use of antibodies within an assay system.

  The properties of zinc fingers, zinc finger motifs, and their interactions are described in Nardelli et al. "Zinc finger DNA recognition: analysis of basic specificity by position-directed mutagenesis", Nucleic Acids Res. , 20 (16): 4137-44 (1992), Jamison et al. "In vitro selection of zinc fingers with altered DNA-binding specificity", Biochemistry, 33 (19): 5687-95 (1994), Chandrasegaran and Smith, "Chimeric restriction enzyme: what's next", Biol Chem 380 (7-8): 841-8 (1999), and Smith et al. "Detailed Study of Substrate Specificity of Chimeric Restriction Enzymes", Nucleic Acids Res. 27 (2): 674-81 (1999).

  One type of specific binding molecule is an oligonucleotide or oligonucleotide derivative. The specific binding molecule is designed and used to detect a specific nucleic acid sequence. Thus, the analyte for the oligonucleotide specific binding molecule is a nucleic acid sequence. The analyte may be a nucleotide sequence within a larger nucleic acid molecule. Oligonucleotide-specific binding molecules can have any length that supports the unique and stable hybridization between multidimensional molecules and analytes. For this purpose, a length of 10-40 nucleotides is preferred, and an oligonucleotide specific binding molecule is most preferred of a length of 16-25 nucleotides. The oligonucleotide specific binding molecule is preferably a peptide nucleic acid. Peptide nucleic acids form stable hybrids with DNA. This allows the peptide nucleic acid specific binding molecule to be firmly attached to the target sequence during subsequent amplification and detection operations.

This effective effect is described in Gasparro et al. Nucleic Acids Res. 22 (14): 2845-2852 (1994), can be obtained with oligonucleotide-specific binding molecules. Briefly, oligonucleotide specific binding molecules are designed to form a triple helix when hybridized to a target sequence. This is accomplished, as is generally known, preferably by selecting either the primary homopurine or the primary homopyrimidine target sequence. The matching oligonucleotide sequences that make up the specific binding molecule complement the selected target sequence and are thus primary homopyrimidines or primary homopurines, respectively. Specific binding molecules (corresponding to the triple helix probe described in Gasparro et al.) Include psoralen derivatives that are chemically linked. After hybridization of the specific binding molecule to the target sequence, a triple helix is formed. By illuminating the triple helix with low wavelength ultraviolet light, the psoralen derivative mediates the cross-linking of the probe to the target sequence.
H. Multidimensional signal fusion A multidimensional signal fusion is a multidimensional signal peptide (ie, a fusion protein) that is joined to the protein or peptide in a single amino acid segment. The above fusion of the protein and peptide with a multidimensional signal peptide can be expressed as a fusion protein or peptide from a nucleic acid molecule that encodes the amino acid segments that make up the fusion. A multidimensional signal fusion nucleic acid molecule or multidimensional signal nucleic acid segment is referred to as a nucleic acid molecule or nucleic acid sequence that encodes a multidimensional signal fusion, respectively.

  The multidimensional signal peptides and proteins included in the multidimensional signal fusion need not be directly fused. That is, other amino acids, amino acid sequences, and / or peptide elements can intervene. For example, if there is an epitope tag, it can be placed between the protein in the multidimensional signal fusion and the multidimensional signal peptide. The multidimensional signal peptide can be fused to the protein in any configuration, such as at the N-terminus of the protein, at the C-terminus of the protein, within the domain junction or at the junction, or at any other suitable location within the protein. Depending on the format of the method, it may be desirable for the protein to be functional. In such cases, terminal fusion or internal domain fusion is desirable. Those skilled in the art of protein fusion are generally aware of how to design a fusion when the protein is functional. In another embodiment, the protein need not be functional if the multidimensional signal peptide and protein have any desired structural organization.

  A given multidimensional signal fusion can include one or more multidimensional signal peptides and one or more proteins or peptides of interest. Furthermore, multidimensional signal fusions can include one or more amino acids, amino acid sequences, and / or peptide elements. The disclosed multidimension signal fusion comprises a single, continuous polypeptide chain. Thus, a plurality of amino acid segments may be part of the same continuous polypeptide chain, but all components of a given amino acid segment (ie, the relevant multidimensional signal peptide, protein, and peptide) are the same Part of a continuous polypeptide chain.

  In a preferred embodiment, a nucleic acid comprising a multidimensional signal peptide, a multidimensional signal fusion (or amino acid segment), a nucleic acid segment encoding a multidimensional signal fusion, and / or a nucleic acid segment encoding a multidimensional signal fusion. Molecules are sets in which a multidimensional signal peptide, multidimensional signal fusion, and / or subsegments of a multidimensional signal fusion have similar properties (such as similar mass to charge ratios) that make up or are present in the set. used. Similar properties can distinguish and / or separate multidimension signals, multidimension signal fusions, or subdimensions of multidimension signal fusions from other molecules that lack one or more properties. Preferably, the subdimensions of a multidimensional signal, multidimensional signal fusion, or multidimensional signal fusion that comprise or are present in the set have the same mass to charge ratio (m / z). That is, the multi-dimensional signal, multi-dimensional signal fusion, or sub-segment of the multi-dimensional signal fusion in the set may be isobaric. This allows multidimensional signals, multidimensional signal fusions, or sub-segments of multidimensional signal fusions to be accurately separated from other molecules based on mass to charge ratio. As a result of filtering, the signal-to-noise ratio (S / N) for the system is greatly increased, allowing for more sensitive and accurate detection.

  Multiple sets of multidimensional signal fusions (also referred to as amino acid segments), multidimensional signal fusion fragments (also referred to as subsegments or amino acid subsegments of multidimensional signal fusions), multidimensional signal peptides, multidimensional signal fusions A nucleic acid molecule comprising a nucleic acid segment that encodes or a nucleic acid segment that encodes a multidimensional signal fusion encodes any number of multidimensional signal fusions, multidimensional signal fusion fragments, multidimensional signal peptides, multidimensional signal fusions Nucleic acid molecules can be included that comprise nucleic acid segments or nucleic acid segments that encode multidimensional signal fusions. For example, a nucleic acid molecule comprising multiple sets of multidimensional signal fusions, multidimensional signal fusion fragments, multidimensional signal peptides, nucleic acid segments that encode multidimensional signal fusions, or nucleic acid segments that encode multidimensional signal fusions, 1, 2, or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, or 500 or more different multidimensional signal fusion, multidimensional signal fusion fragment, multidimensional signal peptide, nucleic acid segment encoding multidimensional signal fusion, Alternatively, it can have a nucleic acid molecule comprising a nucleic acid segment that encodes a multidimensional signal fusion. Nucleic acid molecules comprising a specific number of multidimensional signal fusions, multidimensional signal fusion fragments, multidimensional signal peptides, nucleic acid segments encoding multidimensional signal fusions, and nucleic acid segments encoding multidimensional signal fusions, and A multidimensional signal fusion, a multidimensional signal fusion fragment, a multidimensional signal peptide, a nucleic acid segment encoding a multidimensional signal fusion, and a specific endpoint for a range of nucleic acid molecules comprising a nucleic acid segment encoding a multidimensional signal fusion A listed number of multidimensional signal fusions, multidimensional signal fusion fragments, multidimensional signal peptides, nucleic acid segments encoding multidimensional signal fusions, and nucleic acid segments encoding multidimensional signal fusions Nucleic acid molecules, multiple multidimensional signal fusions, multidimensional signals Specific fusion endpoints, multidimensional signal peptides, nucleic acid segments encoding multidimensional signal fusions, and each specific endpoint for a range of nucleic acid molecules comprising nucleic acid segments encoding multidimensional signal fusions A specific number of multidimension signal fusions, multidimension signal fusion fragments, multidimension signal peptides, nucleic acid segments that encode multidimension signal fusions, and multidimension signal fusions that are intended and not specifically listed Each nucleic acid molecule comprising a nucleic acid segment to be encoded and a number of multidimensional signal fusions, multidimensional signal fusion fragments, multidimensional signal peptides, nucleic acid segments encoding multidimensional signal fusions, and multidimensional signal fusions Specific endpoints for a range of nucleic acid molecules comprising a nucleic acid segment encoding One one has been specifically described by the specification.

  Multidimensional signal fusions can be expressed in any suitable manner. For example, nucleic acid sequences encoding nucleic acid sequences and multidimensional signal fusions can be expressed in vitro, in cells, and / or in cells of living organisms. Many techniques and systems for the expression of nucleic acid sequences and proteins are known and can be used with the disclosed multidimensional signal fusions. For example, many expression sequences, vector systems, conversion and transfer techniques, and genetically engineered organism production methods are known and can be used with the disclosed multidimensional signal peptide methods and compositions. Systems that integrate nucleic acid constructs with cellular and biological chromosomes are known (eg, Groth et al. (2000) “Phage integrase leads to efficient site-specific integration in human cells”, ProcNatlAcadSciUSA97: 5995. Hong et al. (2001) A set of large genes in mammalian cells that can be used with the disclosed nucleic acid molecules and segments that encode multidimensional signal fusions, or that form nucleic acid segments that encode multidimensional signal fusions. Development of two bacterial artificial chromosome shuttle vectors for recombination-based cloning and regulated expression ", Analytical Biochemistry 291: 142-148)).

  As used herein, an expression sample is a sample that contains or can contain one or more multidimensional signal fusions expressed from a nucleic acid molecule. The analyzed expression sample can be fractionated or separated to reduce sample complexity. Fragmentation and fractionation can also be used together in the same assay. The fragmentation and fractionation can simplify and extend the analysis of expression.

  A nucleic acid molecule that encodes a multidimensional signal fusion is such that the multidimensional signal peptide within the multidimensional signal fusion encoded by a set of nucleic acid molecules separates or distinguishes the multidimensional signal peptide from molecules lacking common properties. Can be used in a set that can have one or more possible common characteristics. Similarly, a nucleic acid molecule that encodes an amino acid segment is such that a multidimensional signal peptide within an amino acid segment encoded by a set of nucleic acid molecules separates or distinguishes the multidimensional signal peptide from molecules lacking common properties. It can be used in a set that can have one or more possible common characteristics. A nucleic acid molecule that encodes an amino acid segment has one or more common characteristics that allow the amino acid segment encoded by a set of nucleic acid molecules to separate or distinguish the amino acid segment from molecules that lack common characteristics Can be used in a set of

  Similarly, a nucleic acid molecule that encodes a multidimensional signal fusion includes one or more multidimensional signal peptides within the multidimensional signal fusion encoded by a set of nucleic acid molecules that generate a pattern in indicator level analysis. It can be used in a set that can have properties. Similarly, a nucleic acid segment that encodes a multidimensional signal fusion (generally part of a nucleic acid molecule) is a multidimensional signal peptide within the multidimensional signal fusion encoded by a set of nucleic acid segments that produces a pattern. It can be used in a set that can have one or more characteristics. Consider multiple sets of nucleic acid molecules, nucleic acid segments, amino acid segments, multidimensional signal peptides, and other relationships between members of the protein.

A nucleic acid segment that encodes a multidimensional signal fusion (generally part of a nucleic acid molecule) is a multidimensional signal peptide within a multidimensional signal fusion that is encoded by a set of nucleic acid segments. Can be used in sets that can have one or more common properties that can be separated or distinguished from molecules lacking. Similarly, a nucleic acid segment encoding an amino acid segment is one in which a multidimensional signal peptide within an amino acid segment encoded by a set of nucleic acid molecules separates or distinguishes the multidimensional signal peptide from molecules lacking common properties. It can be used in a set that can have one or more possible common characteristics. A nucleic acid segment that encodes an amino acid segment has one or more common characteristics that allow the amino acid segment encoded by a set of nucleic acid molecules to separate or distinguish the amino acid segment from molecules that lack common characteristics. It can be used in a set that can. Consider multiple sets of nucleic acid molecules, nucleic acid segments, amino acid segments, multidimensional signal peptides, and other relationships between members of the protein.
I. Multidimensional signal / analyte conjugate The composition in which a multidimensional signal is associated with, incorporated into, or otherwise bound to an analyte or protein is a multidimensional signal / analyte conjugate (or MDS / analyte conjugate) or multiple. Dimensional signal / protein conjugate (or MDS / protein conjugate). The conjugate includes a multidimensional signal associated with a specimen such as a multidimensional signal probe mixed with a nucleic acid sequence, a multidimensional signal covalently bound to a specimen such as a multidimensional signal linked to a protein via a linking group, And multidimensional signals that are incorporated into analytes such as fusions between the protein and multidimensional signal peptides (or peptide multidimensional signals).

  Depending on the embodiment of the disclosed method that employs multidimensional signals, the multidimensional signals can be varied and different forms of different multidimensional signals can be distinguished from one another. The multidimension signal / analyte conjugate can be altered, generally by changing the multidimension signal portion of the conjugate, resulting in a different form of a different multidimensional signal, an altered form of a different multidimensional signal / analyte conjugate, or Both can be distinguished from each other. If the multidimension signal or multidimension signal / analyte conjugate is altered by fragmentation, some or all of the fragments can be distinguished from each other in some embodiments. For example, if a multidimensional signal is fragmented into two parts, one or both parts of the multidimensional signal can be distinguished. When a multidimensional signal / analyte conjugate is fragmented into two parts (with a break in the multidimensional signal part), it can distinguish between multidimensional signal fragments, multidimensional signal / analyte fragments, or both it can. In some embodiments, only one part of the fragmented multidimensional signal can be detected, so only this part of the reporter signal needs to be distinguished.

 Multiple sets of multidimension signal / analyte conjugates are those in which two or more multidimension signal / analyte conjugates in a set distinguish multidimensional signal / analyte conjugates that have common properties from molecules that lack other common properties and It can be used if it has one or more common properties that can be separated. In yet another embodiment, the analyte can be fragmented (before or after binding) to create a multidimensional signal / analyte fragment conjugate (also referred to as a fragment conjugate). In such a case, a set of fragment conjugates allows two or more fragment conjugates in the set to distinguish and / or separate fragment conjugates having common properties from molecules lacking other common properties. It can be used when it has more than one common characteristic. It should be understood that a fragmented specimen can itself be considered a specimen. In view of this, references to fragmented samples are made for clarity in describing specific embodiments for convenience and may be considered as references to both base and fragmented samples. it can.

  Multiple sets of multidimensional signal / analyte conjugates or multidimensional signal / analyte fragment conjugates (fragment conjugates) can have any number of multidimensional signal / analyte conjugates or multidimensional signal / analyte fragment conjugates. . For example, multiple sets of multidimension signal / analyte conjugates or multidimension signal / analyte fragment conjugates are one, two or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, or 500 or more different multidimensional signals / It can have an analyte conjugate or a multidimensional signal / analyte fragment conjugate. A specific number of multidimension signal / analyte conjugates and multidimension signal / analyte fragment conjugates, and a number of specific end points in a range of multidimension signal / analyte conjugates and multidimension signal / analyte fragment conjugates are listed. A specific number of multidimension signal / analyte conjugates and each one of the multidimension signal / analyte fragment conjugates, and specific endpoints for a range of multiple multidimension signal / analyte conjugates and multidimension signal / analyte fragment conjugates Each of which is specifically intended and not specifically listed, but each of a specific number of multidimensional signal / analyte conjugates and multidimensional signal / analyte fragment conjugates, and a number of Each one of the specific endpoints of the range of multidimensional signal / analyte conjugates and multidimensional signal / analyte fragment conjugates is thereby specifically described.

  As described above, the multidimensional signal that is bound to the analyte or protein can be altered and distinguished while in the conjugate. The bound multidimensional signal can be partially or fully dissociated or separated from the bound analyte prior to modification. Other bound multidimensional signals can also be partially or fully dissociated or separated from the bound analyte prior to analysis. If the multidimensional signal is dissociated (partially or fully) from the sample, the method is performed so that the association between the sample and the multidimensional signal is part of the information obtained when the multidimensional signal is detected can do. That is, the fact that the multidimensional signal can be dissociated from the specimen for detection does not obscure the information that the detected multidimensional signal has been associated with the specimen.

  As used herein, bound multidimensional signal conjugates refer to both multidimensional signal / analyte conjugates and other components of the disclosed methods, such as multidimensional molecules.

Similar to multidimensional signals, generally multidimensional signal / analyte conjugates and multidimensional signal / analyte fragment conjugates are multidimensional signal / analyte conjugates or fragment conjugates in a set that are multidimensional signal / analyte conjugates. Alternatively, fragment conjugates can be used in sets that have one or more common properties that can be separated or distinguished from molecules lacking common properties.
J. et al. Capture Array A capture array (also referred to herein as an array) includes a plurality of capture tags that are secured to a solid substrate, preferably at a particular or predetermined location on the solid substrate. In this situation, multiple capture tags refer to multiple capture tags each having a different structure. Preferably, a given location on the array (also referred to herein as an array element) has one type of capture tag (ie, all capture tags at that location have the same structure). Each location has a plurality of duplicate capture tags. Spatial separation of differently structured capture tags in the array individually detects and identifies the analyte associated with the capture tag. If a decoding tag is detected at a given position in the capture array, it indicates that the analyte corresponding to that array element was present in the target sample.

  The solid substrate used in the capture array can include any solid material to which the capture tag is directly or indirectly bound. This includes acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicate, polycarbonate, teflon, fluorocarbon, nylon, silicone rubber, polyanhydride Materials such as polyglycolic acid, polylactic acid, polyorthoesters, polypropyl fumerate, collagen, glycosamminglecan, and polyamino acids. The solid substrate can take any effective form such as a thin film or membrane, beads, bottles, dishes, disks, compact disks, fibers, optical fibers, woven fibers, molded polymers, particles and particulates. A preferred shape for a solid substrate is a compact disc.

  Although preferred, a given capture array need not be a single unit or structure. The set of capture tags can be distributed on any number of solid supports. For example, in extreme cases, each capture tag can be secured to a separate reaction tube or vessel. The array may be constructed on an impermeable or permeable carrier of a wide range of carrier compositions as described above. The spot size and spot packing density of the array vary over a very wide range depending on the process and material used.

  Methods for immobilizing antibodies and other proteins on a substrate are well established. Immobilization can be accomplished, for example, by attachment to aminated, carboxylated, or hydroxylated surfaces using standard immobilization chemistries. Examples of binders are brominated cyanide, succinimide, aldehyde, tosyl chloride, avidin-biotin, photocrosslinker, epoxide, and maleimide. A preferred binder is glutaraldehyde. The methods used in conjugation with these and other binding agents are described in “Protein Immobilization: Fundamentals and Applications”, Richard F. et al. Edited by Taylor (M. Dekker, New York, 1991), Johnston and Thorpe, "Practical Immunochemistry" (Blackwell Scientific Publications, Oxford, England, 1987) 209-216 and 241-242 Ligand, Affinity Pages. T.A. Hermanson et al. (Academic Press, New York, 1992). The antibody can be attached to the substrate by a chemically cross-linked free amino group on the antibody to a reactive side group present in the substrate. For example, antibodies can be chemically cross-linked to substrates containing free amino groups or carboxyl groups using glutaraldehyde or carbodiimide as a cross-linking agent. According to this method, an aqueous solution containing free antibodies is incubated with a solid substrate in the presence of glutaraldehyde or carbodiimide. For crosslinking with glutaraldehyde, the reactants can be incubated with 2% mass glutaraldehyde in a lysis buffer such as 0.1 M sodium cacodylate at pH 7.4. Other standard fixed chemicals are known by those skilled in the art.

  Methods for immobilizing oligonucleotides to a solid substrate are well established. The oligonucleotide capture tag can be linked to the substrate using established binding methods. For example, a suitable addition method is described by Pease et al. Proc. Natl. Acad. ScL, USA 91 (ll): 5022-5026 (1994), Krapko et al. MolBiol (Mosk) (USSR) 25: 718-730 (1991), Fodor et al. U.S. Pat. No. 5,871,928, Brenner U.S. Pat. No. 5,654,413, U.S. Pat. No. 5,429,807, and Pease et al. U.S. Pat. No. 5,599,695. Methods for immobilizing 3'-amine oligonucleotides on casein-coated slides are described in Stimpson et al. Proc. Natl. Acad. ScL, USA 92: 6379-6383 (1995). A suitable method for adding oligonucleotides to a solid substrate is described by Guo et al. Nucleic Acids Res. 22: 5456-5465 (1994).

  Planar array technology has been utilized for many years (Shalon, D., SJ Smith, and PO Brown, “DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization”). Genome Res, 1996, 6.6 (7): p. 639-45, Singh-Gasson, S., et al., “Masked-less production of light-directed oligonucleotide microarrays using digital micromirror arrays”, Nat Biotechnol. 1999. 17 (10): p. 974-8, Southern, EM, U. Maskos, and JK Elder, “Analysis and comparison of nucleic acid sequences by hybridization of oligonucleotides to an array: Evaluation using experimental models ", Ge nomics, 1992.13 (4): p. Proc Natl Acad Sci USA, 1991.88 (8): p. 3233-7, Van Oss, CJ., RJ. Good, and MK Chaudhury, “Cellulose nitrate and other membrane DNA (Southern) and proteins ( Western) Blotting Mechanism ", J Chromatogr, 1987. 391 (1): p. 53-65, Ramsay, G.," DNA chip: state of the art ", Nat Biotechnol, 1998.16 (1): p. ~ 4, Schena, M., et a , "Parallel human genome analysis: microarray-based expression monitoring of 1000 genes", ProcNatlAcadSciUSA, 1996.93 (20): p.10614-9, Lipshutz, RJ., Et al., "High-density synthetic oligonucleotide arrays", Nat Genet, 1999.21 (1 Suppl): p.20-4, Pease, AC, et al., “Luminescent Oligonucleotide Arrays for Rapid DNA Sequence Analysis”, ProcNatlAcadSciUSA, 1994.91 (11): p. 5022-6, Maier, E. et al., "Application of robotic techniques to automated sequence fingerprint analysis by oligonucleotide hybridization", J Biotechnol, 1994.35 (2 To 3): p. 191-203, Vasiliskov, A.M. V. Et al. "Manufacturing a microarray of gel-immobilized compounds on a chip by copolymerization," Biotechniques, 1999. 27 (3): p. 592-4, 596-8, 600passim, and Yershov, G .; Et al. "DNA analysis and diagnosis of oligonucleotide microchips," ProcNatlAcadSciUSA, 1996. 93 (10): p. 4913-8).

  The oligonucleotide capture tags in the array can also be designed to have similar hybrid stability. Thereby, the hybridization of fragments to the capture tag is made more efficient, and the occurrence of hybridization mismatches is reduced. Hybrid stability of oligonucleotide capture tags can be calculated using known formulas and thermodynamic principles (eg, Santa Lucia et al., Biochemistry 35: 3555-3562 (1996); Freier et al., Proc. Natl. Acad. Sci. USA 83: 9373-9377 (1986); Breslauer et al., Proc. Natl. Acad. Sci. USA 83: 3746-3750 (1986)). Hybrid stability of oligonucleotide capture tags can be made more similar, for example, by chemically modifying the capture tag (a process referred to as facilitating hybrid stability) (Nguyen et al., Nucleic Acids). Res.25 (15): 3059-3065 (1997); Hohsisel, Nucleic Acids Res.24 (3): 430-432 (1996)). Hybrid stability can also be facilitated by performing hybridization under special circumstances (Nguyen et al., Nucleic Acids Res. 27 (6): 1492-1498 (1999); Woodet al., Proc. Natl.Acad.Sci.USA82 (6): 1585-1588 (1985)).

  Another means of facilitating the hybrid stability of the oligonucleotide capture tag is to vary the length of the capture tag. This allows the hybrid stability of each capture tag to be adjusted so that all capture tags can have similar hybrid stability (as much as possible). It is understood that the hybrid stability of the capture tag in the capture array is not uniform because the addition or deletion of a single nucleotide to or from the capture tag changes the capture tag's hybrid stability in fixed increments. Thus, the hybrid stability similarity, as used herein, increases the similarity of the capture tag hybrid stability (or, in other words, the difference in the capture tag hybrid stability difference). Reduction).

Hybridization efficiency and ligation of oligonucleotide capture tags to sample fragments is also improved by grouping capture tags with similar hybrid stability at a site or segment of the capture array that can be subjected to different hybridization conditions. be able to. In this way, the hybridization conditions can be optimized for a particular class of capture tags.
K. Capture tag A capture tag is a compound that can be used to capture or separate a compound or complex having a capture tag. Preferably, the capture tag is a compound that interacts specifically with a particular molecule or moiety. Preferably, the molecule or moiety that interacts specifically with the capture tag is an analyte. The term analyte is understood to refer to both a distinct molecule and a part of a molecule, such as an epitope of a protein, that specifically interacts with a capture tag. Antibodies of either receptor / ligand pairs or synthetic polyamides (Dervan and Burli, “Sequence with Polyamide—Specific DNA Recognition”, Curr Opin Chem Biol, 3 (6): 688-93 (1999); Wemmer and Dervan , “Targeting the DNA minor groove”, Curr Opin Struct Biol, 7 (3): 355-61 (1997)), nucleic acid probes, and other molecules with specific binding affinity are examples of capture tags.

  A capture tag that interacts specifically with a particular analyte is said to be specific to that analyte. For example, if a capture tag is an antibody that is associated with a particular antigen, the capture tag is said to be specific to that antigen. An antigen is a specimen. The capture tag is preferably an antibody, ligand, binding protein, receptor protein, hapten, aptamer, carbohydrates, synthetic polyamides, peptide nucleic acids, or oligonucleotides. A preferred binding protein is a DNA binding protein. Preferred DNA binding proteins are zinc finger motifs, leucine zipper motifs, helix-turn-helix motifs. These motifs can be combined with the same capture tag.

  Antibodies useful as affinity portions of multidimensional molecules are commercially available or can be made using well-established methods. For example, Johnston and Thorpe, "Practical Immunochemistry" (Blackwell Scientific Publications, Oxford, England, 1987), pages 30-85, for general methods effective in creating both polyclonal and monoclonal antibodies. Says. The entire book describes many general techniques and principles for the use of antibodies within an assay system.

  The properties of zinc fingers, zinc finger motifs, and their interactions are described in Nardelli et al. "Zinc finger DNA recognition: analysis of basic specificity by position-directed mutagenesis", Nucleic Acids Res. , 20 (16): 4137-44 (1992), Jamison et al. , "In vitro selection of zinc fingers with altered DNA-binding specificity", Biochemistry, 33 (19): 5687-95 (1994), Chandrasegaran and Smith, "Chimeric restriction enzyme: what's next", Biol Chem, 380 (7-8): 841-8 (1999), and Smith et al. "Detailed Study of Substrate Specificity of Chimeric Restriction Enzymes", Nucleic Acids Res. 27 (2): 674-81 (1999).

  One type of capture tag is an oligonucleotide or oligonucleotide derivative. The capture tag is designed and used to detect a specific nucleic acid sequence. Thus, the analyte for the oligonucleotide capture tag is a nucleic acid sequence. The analyte may be a nucleotide sequence within a larger nucleic acid molecule. The oligonucleotide capture tag can be of any length that supports a specific stable hybridization between the capture tag and the analyte. For this purpose, a length of 10-40 nucleotides is preferred, and an oligonucleotide capture tag with a length of 16-25 nucleotides is most preferred. The oligonucleotide capture tag is preferably a peptide nucleic acid. Peptide nucleic acids form stable hybrids with DNA. Thus, the peptide nucleic acid capture tag is firmly attached to the target sequence during subsequent amplification and detection operations.

This beneficial effect is described in Gasparro et al. Nucleic Acids Res. 22 (14): 2845-2852 (1994), can be obtained with oligonucleotide capture tags by utilizing the triple helix chemical coupling technique. Briefly, oligonucleotide capture tags are designed to form a triple helix when hybridized to a target sequence. This is accomplished, as is generally known, preferably by selecting either the primary homopurine or the primary homopyrimidine target sequence. The matching oligonucleotide sequence that constitutes the capture tag complements the selected target sequence, thus becoming a primary homopyrimidine or primary homopurine, respectively. Supplementary tags (corresponding to the triple helix probe described in Gasparro et al.) Include psoralen derivatives that are chemically coupled. After hybridization of the supplementary tag to the target sequence, a triple helix is formed. By illuminating the triple helix with low wavelength ultraviolet light, the psoralen derivative mediates the cross-linking of the probe to the target sequence.
L. Sample Array The sample array includes a plurality of samples (eg, expression samples, tissue samples, protein samples) fixed to a solid substrate, preferably at a specific position or a predetermined position on the solid substrate. Preferably, each predetermined location on the sample array (referred to herein as a sample array element) has one type of sample. Spatial separation of different samples in a sample array to separate multidimensional signals (or multidimensional molecules, multidimensional signals, multidimensional molecules, indicator signals, indicator molecules, or encoding tags) associated with a sample Can be detected and identified. If a multidimensional signal is detected at a given position in the sample array, it indicates that an analyte corresponding to the multidimensional signal is present in the sample corresponding to the sample array element.

  The solid substrate used in the sample array can include any solid material to which a sample can be directly or indirectly attached. This includes acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicate, polycarbonate, teflon, fluorocarbon, nylon, silicone rubber, polyanhydride. Materials such as polyglycolic acid, polylactic acid, polyorthoesters, polypropyl fumerate, collagen, glycosamminglecan, and polyamino acids. The solid substrate can take any effective shape such as a thin film or membrane, beads, bottles, dishes, disks, compact disks, fibers, optical fibers, woven fibers, molded polymers, particles, and particulates. The preferred shape of the solid substrate is a compact disc.

  Although preferred, a given sample array need not be a single unit or structure. The set of samples can also be distributed on any number of solid supports. For example, in extreme cases, each sample can be fixed in a separate reaction tube or vessel. The sample array may be constructed on an impermeable or permeable carrier of a wide range of carrier compositions as described above. The spot size and spot packing density of the array vary over a very wide range depending on the process and material used. Methods for attaching or fixing samples and sample components to a substrate have been established.

A preferred shape for the sample array is a tissue array with a small tissue sample on the substrate. The tissue microarray exists, for example, in a cohort for studying breast cancer and is used there. The disclosed method can be used, for example, to probe multiple analytes in multiple samples. The sample array is labeled, for example, with different multidimensional signals, after which the entire support is introduced into the source region of mass properties and sampled by MALDI.
M.M. Decoding tag A decoding tag is any molecule or moiety that can be directly or indirectly associated with an encoding tag. The decoding tag is associated with a multidimensional signal (constituting a multidimensional molecule) that can indirectly associate the multidimensional signal with the specimen. Decoding tags are preferably oligonucleotides, carbohydrates, synthetic polyamides, peptide nucleic acids, antibodies, ligands, proteins, haptens, zinc fingers, aptamers, or mass labels.

  Suitable decoding tags are molecules that can be hybridized specifically to oligonucleotide coding tags. Most preferred is a peptide nucleic acid decoding tag. The oligonucleotide or peptide nucleic acid decoding tag can have any sequence. The only requirement is hybridization to the encoding tag. Each decoding tag can have any length that supports a particular stable hybridization between the encoding tag and the decoding tag. For this purpose, a length of 10-35 nucleotides is preferred, and a decoding tag is most preferred a length of 15-20 nucleotides.

Multidimensional molecules containing decoding tags can preferably be released by matrix-assisted laser desorption ionization (MALDI) or another detection technique for separation and identification by time of flight (TOF) mass spectrometry. The decoding tag may be an oligomer molecule that can be hybridized to the encoding tag. For example, the decoding tag may be a DNA oligonucleotide, an RNA oligonucleotide, or a peptide nucleic acid (PNA) molecule. A preferred decryption tag is a PNA molecule.
N. Encoding tag An encoding tag is a molecule or moiety that can be associated with a decoding tag. The encoding tag may be any type of molecule or moiety that can serve as a target for decoding tag association. Suitable coding tags are oligomers, oligonucleotides or nucleic acid sequences. The encoding tag may be a member of a binding pair such as streptavidin or biotin and its cognate decoding tag is another member of the binding pair. Encoding tags can also be designed to be directly associated with several types of multidimensional signals. For example, oligonucleotide coding tags can be designed to interact directly with peptide nucleic acid multidimensional signals (multidimensional signals consisting of peptide nucleic acids).

  The oligomer-based sequence of the oligomer-encoded tag comprises RNA, DNA, modified RNA or DNA, modified backbone nucleotide oligomers, such as peptide nucleic acids, methylphosphonate DNA, and 2′-0-methyl RNA or DNA. Can be included. The oligomer or oligonucleotide encoding tag can have any sequence. The only requirement is an association with the decryption tag (preferably by hybrid). In the disclosed method, multiple encoding tags can be associated with a single analyte. The context of these multiple encoding tags depends on the technology used for signal amplification. Thus, when branched DNA is used, the branched DNA molecule includes multiple coding tags on the branch. If oligonucleotide dendrimers are used, the coding tag is on the dendrimer arm. If rotating circle replication is used, the plurality of encoding tags result from tandem repeats of the complement of the amplified target circle sequence (including at least one complement of the encoding tag sequence). In this case, the encoding tag is repeated in a single row with tandem sequence DNA.

  Each of the oligonucleotide coding tags can have any length that supports a unique and stable hybridization between the coding tag and the decoding tag. For this purpose, a length of 10-35 nucleotides is preferred, and coding tags are most preferred 15-20 nucleotides in length.

  Branched DNA used in the disclosed methods is generally known (Urdea, Biotechnology 12: 926-928 (1994), and Horn et al., Nucleic Acids Res. 23: 4835-4841 (1997)). As used herein, the tail of a branched DNA molecule refers to the part of the branched DNA molecule that is designed to interact with the analyte. The tail is a specific binding molecule. In general, each branched DNA molecule should have only one tail. A branch of branched DNA (also referred to herein as an arm of the branched DNA) contains a coding tag sequence. Oligonucleotide dendrimers (or dendrimer DNA) are also generally known (Shchepinov et al., Nucleic Acids Res. 25: 4447-4454 (1997), and Orentas et al., J. Virol. Methods 77: 153-163 ( 1999)). As used herein, the tail of an oligonucleotide dendrimer refers to the part of a dendrimer that is designed to interact with an analyte. In general, each dendrimer should have only one tail. The dendrimer strand of a dendrimer is referred to herein as the arm of an oligonucleotide dendrimer and includes a coding tag sequence.

A coding tag can link an analyte or another molecule to be labeled (directly or via a linker or spacer). The encoding tag can also be associated with the analyte to be labeled and another molecule. For this purpose, encoded molecules are preferred. An encoded molecule is a molecule that can interact with an analyte and a decoding tag. The encoding molecule includes a specific binding molecule and an encoding tag. The specific binding molecule is as described above.
O. Multidimensional carrier and encoded carrier A multidimensional carrier is an association of one or more specific binding molecules, carriers, and multiple multidimensional signals. Multidimensional carriers are used in the disclosed methods to associate multiple multidimensional signals with analytes. A coded carrier is an association of one or more specific binding molecules, a carrier, and multiple coding tags. The encoded carrier is used in the disclosed method to associate multiple encoded tags with analytes. A carrier may be a molecule or structure that simplifies the association of many multidimensional signals with specific binding molecules. For example, liposomes, microparticles, nanoparticles, virons, phagemids, and branched polymer structures. The general class of carriers are structures or materials designed for drug delivery. Many such carriers are known. Liposomes are a suitable form of carrier.

  Liposomes are artificial structures mainly composed of phospholipid bilayers. Cholesterol and fatty acids can be included in the bilayer structure. Depending on the format of the disclosed method, the liposome serves as a carrier for any multidimensional signal or encoding tag. A high multiplex assay can be performed by combining a liposome multidimensional carrier loaded with any signal or tag and a method capable of separating very broad overlaps of signal and tag.

  Liposomes, preferably unilamellar vesicles, are created using established procedures that lead to the loading of internal compartments with numerous (thousands) multidimensional signals or encoded tag molecules, and the chemistry of these molecules Is suitable for submission by preselected detection methods. A particular type of multidimensional signal or encoding tag is preferably used for each particular type of liposome carrier.

  Each specific type of liposomal multidimensional or encoded carrier is associated with a specific binding molecule. The association may be direct or indirect. An example of direct binding is a liposome containing an antibody covalently bound to the surface of a phospholipid bilayer. Another indirect binding composition is a liposome containing any sequence of DNA oligonucleotides covalently bound to the surface. These oligonucleotides are designed to recognize specific multidimensional molecules by base complementarity. A multidimensional molecule can comprise an antibody-DNA covalent complex, whereby the DNA portion of the complex can be clearly hybridized with complementary sequences on a liposomal multidimensional carrier. In this way, liposomal multidimensional carriers become common reagents that can be indirectly associated with any desired binding molecule.

  The use of liposomal multidimensional carriers can be illustrated by the following example.

  1. Liposomes (preferably monolayer vesicles with an average diameter of 150-300 nanometers) are made using extrusion methods (Hope et al., Biochimica et Biophysica Acta, 812: 55-65 (1985); MacDonald et al. Biochimica et Biophysica Acta, 1061: 297-303 (1991)). Other methods of liposomal formulation can also be used.

  2. This specific oligopeptide is loaded into the internal volume of the liposome, and a solution of the oligopeptide is used in the liposome formulation at a concentration of 400 micromolar to serve to identify the specific analyte. Liposomes with an inner diameter of 200 nanometers contain an average of 960 molecules of oligopeptide. Three separate liposome formulations are extruded, each loaded with a different oligopeptide. Oligopeptides are selected to be divided into fragments having the same mass-to-charge ratio but different mass-to-charge ratios so that they can be easily separated by mass spectrometry.

  3. The outer surfaces of the three liposome formulations are bound to specific antibodies as follows. a) the first liposomal formulation reacts with an antibody specific for p53 tumor suppressor; b) the second liposomal formulation reacts with an antibody specific for Bcl-2 tumor protein; c) the second The 3 liposomal formulation reacts with antibodies specific for the Her2 / neu cell membrane receptor. The conjugation reaction is performed using standard procedures for the covalent attachment of antibodies to molecules containing reactive amino groups (Hendrickson et at, Nucleic Acids Research, 23: 522-529 (1995); Hermanson, Bioconjugate techniques, Academic Press, 528-569 (1996); Scheffold et al., Nature Medicine 1: 107-110 (2000)). In the case of liposomes, the reactive amino group is a reactive amino group present at the phosphatidylethanolamine site of the liposome.

  4). In order to associate the liposomes with the corresponding protein antigens present in the fixed tissue, a glass slide holding a standard formaldehyde fixation history site is contacted with a mixture of all three liposome formulations and 30 mM Tris-HCl. , PH 7.6, suspended in a buffer containing 100 mM sodium chloride, 1 mM EDTA, 0.1% bovine serum albumin. After 1 hour incubation, slides are washed twice for 5 minutes with the same buffer (30 mM Tris-HCl, pH 7.6, 100 mM sodium chloride, 1 mM EDTA, 0.1% bovine serum albumin). Slides are dried with a stream of air.

  5. Slides are applied with a thin layer of a matrix solution consisting of 10 mg / ml α-cyano-4-hydroxycinnamic acid, 0.1% trifluoroacetic acid in a 50:50 mixture of acetonitrile and water. Slides are dried with a stream of air.

  6). Slides are placed on the surface of a MALDI plate, and Loboda et al. "MALDI-QqTOF Mass Spectrometer Design and Performance", 47th ASMS Conference, Dallas, Texas (1999), Loboda et al. Rapid Comm. Mass Spectrom. 14 (12): 1047-1057 (2000), Shevchenko et al. Anal. Chem. 72: 2132-1142 (2000), and Kruchinsky et al. J. et al. Am. Soc. Mass Spectrom. 11 (6): 493-504 (2000).

  7. The mass spectrum is obtained from a predetermined position on the slide surface. The relative amount of the three peaks of the multidimensional signal polypeptide is used to determine the relative amount of antigen detected by the liposome-detector complex.

The liposome carrier method is not limited to detection of a specimen on a tissue site. Cells obtained by sorting can also be used for analysis in the disclosed method (Scheffold, A., Assenmacher, M., Reiners-Schramm, L., Lauster, R., and Radbruch, A , 2000, Nature Medicine 1: 107-110).
p. Labeled proteins and analytes Labeled proteins are proteins or peptides to which one or more multidimensional signals are added. Preferably, the multidimensional signal and the protein or peptide are covalently linked or linked together. A labeled sample is a sample to which one or more multidimensional signals are added. Preferably, the multidimensional signal and the analyte are covalently bound or linked together.

  As used herein, molecules are linked when they are covalently bonded, either directly or indirectly. Multidimensional signals can be added to proteins, peptides, or analytes in any way. One non-limiting form of indirect linkage is through a linker molecule. A multidimensional signal can be bound to a protein, peptide, or analyte by any suitable binding reaction. For example, a multidimensional signal can be covalently linked to a protein or peptide by a sulfur-sulfur bond between a cysteine or peptide on the protein and a cysteine on the multidimensional signal. Multidimensional signals can also be added to proteins and peptides by linkage (eg, protein linkage of a multidimensional signal peptide to a protein). Many other chemicals and techniques for binding compounds to proteins, peptides or analytes are known and can be used to bind multidimensional signals to proteins, peptides or analytes. For example, conjugation can be performed using thiols, epoxides, thiols for thiols, NHS esters, isothiocyanates, isothiocyanates for amines, amines, and alcohols for carboxylic acids. Proteins, peptides, and analytes can also be labeled in vivo. As used herein, “labeled protein” refers to both proteins and peptides to which one or more multidimensional signals are added. The term labeled protein refers to both proteins and peptides that are added to intact (eg, unfragmented) multidimensional signals and proteins and peptides that are added to modified (eg, fragmented) multidimensional signals. The latter form of labeled protein is referred to as a fragmented labeled protein. Although the protein portion of a labeled protein can be fragmented (eg, by protease digestion), the term fragmented labeled protein refers to a labeled protein in which a multidimensional signal is fragmented. An isobaric labeled protein is a homologous protein or peptide that is labeled with an isobaric multidimensional signal so that a set of proteins has the same mass to charge ratio.

  As used herein, “labeled analyte” refers to an analyte to which one or more multidimensional signals are added. The term labeled analyte refers to both an analyte that is added to a neat (eg, unfragmented) multidimensional signal and an analyte that is added to a modified (eg, fragmented) multidimensional signal. The latter form of labeled protein is referred to as a fragmented labeled analyte. Although the analyte portion of the labeled analyte can be fragmented, the term fragmented labeled analyte refers to a labeled analyte in which a multidimensional signal is fragmented. Isobaric labeled analytes are homogenous analytes that are labeled with isobaric multidimensional signals such that a set of analytes have the same mass to charge ratio.

  The protein, peptide, or analyte sample to be analyzed can be fractionated or separated to reduce sample complexity. In the same assay, fragmentation and fractionation can be used together. Such fragmentation, splitting, or separation can simplify and extend the analysis of proteins, peptides, and analytes.

In one non-limiting example, a labeled protein can be formed in which a multidimensional signal is specifically added to the phosphorylated peptide. Chemical reactions for specific derivatization of phosphorylin or phosphotyrosine residues have been described (Zhou et al., “A Systematic Approach to the Problem of Protein Phosphorylation”, Nat. Biotech. 19: 375-378 (2001); Oda et al., “Enriched Analysis of Phosphorylated Proteins as a Tool for Testing Phosphoproteomes”, Nat. Biotech 19: 379-382 (2001)). Tryptic peptides processed by either of these two protocols display reactive sulfhydryls at protein phosphorylation sites. These sites can react with multidimensional signals to produce labeled proteins. Non-phosphorylated peptides are not derivatized.
Q. Affinity tag An affinity tag is any compound that can be used to separate a compound or complex having an affinity tag from a compound or complex that does not have an affinity tag. Preferably, the affinity tag is a compound such as a ligand or hapten that is associated with or interacts with another compound such as a ligand binding molecule or antibody. It is also preferred that the interaction between the affinity tag and the capture component, such as between a hapten and an antibody or a ligand and a ligand binding molecule, is a specific interaction. The affinity tag is preferably an antibody, ligand, binding protein, receptor protein, hapten, aptamer, carbohydrates, synthetic polyamides, or oligonucleotides. A preferred binding protein is a DNA binding protein. Suitable DNA binding proteins are zinc finger motifs, leucine zipper motifs, helix-turn-helix motifs. These motifs can be combined with the same specific binding molecule.

  Affinity tags described in the context of nucleic acid probes are described in Syven et al. Nucleic Acids Res. 14: 5037 (1986). Suitable affinity tags include biotin that can be incorporated into nucleic acids. In the disclosed method, the multidimensional signal can be captured, attached or linked to the substrate by an affinity tag incorporated into the multidimensional signal. The capture allows separation of the multidimensional signal from another molecule, which simplifies washing and processing of the multidimensional signal, and automates all or part of the method.

  Zinc fingers can also be used as affinity tags. The properties of zinc fingers, zinc finger motifs, and their interactions are described in Nardelli et al. "Zinc finger DNA recognition: analysis of basic specificity by position-directed mutagenesis", Nucleic Acids Res. , 20 (16): 4137-44 (1992), Jamison et al. "In vitro selection of zinc fingers with altered DNA-binding specificity", Biochemistry, 33 (19): 5687-95 (1994), Chandrasegaran, S .; And J.A. Smith, “Chimeric Restriction Enzymes: What's Next”, Biol Chem 380 (7-8): 841-8 (1999), and Smith et al. "Detailed Study of Substrate Specificity of Chimeric Restriction Enzymes", Nucleic Acids Res. 27 (2): 674-81 (1999).

  Capturing multidimensional signals on a substrate can be performed in several ways, if desired. In certain embodiments, the affinity dock is attached or coupled to the substrate. An affinity dock is a compound or site that mediates the attachment of a multidimensional signal by associating or interacting with an affinity tag on the multidimensional signal. A multidimensional signal on the substrate can be captured by an affinity dock fixed to the substrate. Such capture provides a convenient means of washing away molecules that may interfere with subsequent steps. The captured multidimensional signal can also be released from the substrate. This can be accomplished by separating the affinity tag or breaking the photolabile link between the multidimensional signal and the substrate.

The substrate used in the disclosed method can include any solid material to which a multidimensional signal is attached or bound. Examples of substrates are acrylamide, cellulose, nitrocellulose, glass, silicon, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, teflon, fluorocarbon, nylon, silicone rubber, polyanhydride Including, but not limited to, materials such as ride, polyglycolic acid, polylactic acid, polyorthoester, polypropyl fumerate, collagen, glycosamminglecan, and polyamino acid. The substrate can take any effective form such as a thin film or membrane, beads, bottles, dishes, fibers, optical fibers, woven fibers, molded polymers, particles, compact discs, and particulates.
R. Vectors and expression sequences Gene transfer is not limited to plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, and artificial chromosomes, but may include direct introduction of genetic material therein or cationic liposomes, etc. It can be obtained via transcription of genetic material in a cell or carrier. Such methods are known in the art and can be readily adapted for use in the methods described herein. A transcription vector may be any nucleotide construct used to carry a gene to a cell (eg, a plasmid) or as part of a general strategy for carrying the gene, eg, a part of a recombinant retrovirus or adenovirus. (Ram et al. Cancer Res. 53: 83-88 (1993)). Suitable transfer methods including viral vectors, chemical transfectants, or physicomechanical methods such as electroporation and direct diffusion of DNA are described, for example, in Wolff, J. et al. A., et al. Science, 247, 1465-1468, (1990); and Wolff, J. et al. A. Nature, 352, 815-818 (1991).

  As used herein, a plasmid or viral vector is an agent that transfers a gene to a cell without degradation and includes a promoter that results in the expression of the gene in the transferred cell. In a preferred embodiment, the vector is obtained from either a virus or a retrovirus. Suitable viral vectors are adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, polioviruses, AIDS viruses, neurotrophic viruses, Sindbis or other RNA viruses, including those viruses with an HIV backbone. Any virus family that shares viral properties that make these viruses suitable for use as vectors is also suitable. Suitable retroviruses include murine leukemia virus, MMLV, and retroviruses that express the desired properties of MMLV as a vector. Retroviral vectors are commonly used vectors because they carry larger genetic transport than other viral vectors, ie, carry a transgene or marker gene. However, they are not effective on non-proliferating cells. Adenoviral vectors are relatively stable and easy to function together, have high titers, can be delivered in aerosol compositions, and can transfer non-dividing cells. The poxvirus vector is large and has several sites for inserting genes. They are heat resistant and can be stored at room temperature. A preferred embodiment is a viral vector designed to suppress the host body immune response elicited by viral antigens. A suitable vector of this kind carries the coding region for interleukin 8 or 10.

Viral vectors have a higher processing capacity (ability to introduce genes) than most chemical or physical methods of introducing genes into cells. Viral vectors typically contain nonstructural early genes, structural late genes, RNA polymerase III transcription, inverted terminal repeats necessary for replication and encapsidation, and promoters that control transcription and replication of the viral genome. When designed as a vector, a virus usually has one or more early genes that are removed, and the gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral DNA. This type of structure can carry up to about 8 kb of exogenous genetic material. The necessary function of the removed early gene is usually supplied by a cell line designed to express the gene product of the early gene in trans.
1. Retroviral vectors Retroviruses are animal viruses that belong to the retrovirus virus family of any kind, subfamily, family, or tropism. Retroviral vectors are generally described in Verma, I. et al. M.M. "Retroviral vectors for gene transfer", In Microbiology-1985, American Society for Microbiology, pages 229-232, Washington, (1985). The contents of which are incorporated herein by reference. Examples of methods of using retroviral vectors for gene therapy include US Pat. Nos. 4,868,116 and 4,980,286; PCT applications WO90 / 02806 and WO89 / 07136; and Mulligan, (Science 260: 926-932. (1993)), the teachings of which are incorporated herein by reference.

  Retroviruses are essentially packages that are packed into nucleic acid cargo. The nucleic acid cargo carries a packaging signal with it, thereby ensuring that the replicated daughter molecule is efficiently packaged within the package coat. In addition to the packaging signal, there are a number of molecules that are required in cis for packaging of replicating and replicating viruses. Typically, retroviral genomes contain gag, pol, and env genes that are involved in creating a protein coat. It is the gag, pol, and env genes that are normally replaced by foreign DNA that should be transferred to the target cells. Retroviral vectors usually contain a package signal for incorporation into the package coat, a sequence that signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site for binding a reverse transcription tRNA primer, DNA synthesis A terminal repeat sequence that induces switching of RNA strands in it, a purine-rich sequence 5'-3 'LTR that functions as a priming site for the synthesis of the second strand of DNA synthesis, and a retroviral host in the DNA state Contains specific sequences near the end of the LTR that allow insertion into the genome. By removing the gag, pol, and env genes, approximately 8 kb of foreign sequence can be inserted into the viral genome, reverse transcribed, and packaged into new retroviral particles after replication. This amount of nucleic acid is sufficient to transfer from one gene to many genes, depending on the size of each transcript. It is preferred to include either a positive or negative selectable marker along with other genes in the insert.

Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), vectors are usually generated by placing them in their packaging cell lines. A packaging cell line is a cell line that has been transferred or transformed with a retrovirus that contains the replication and packaging machinery but lacks a packaging signal. When a vector carrying the selected DNA is transferred into these cell lines, the vector containing the gene is replicated and packaged into new retroviral particles by a mechanism provided in cis by helper cells. The genome for the mechanism is not packaged because it lacks the necessary signals.
2. Adenoviral vectors The structure of replication-deficient adenoviruses is described in (Berkner et al., J. Virology 61: 1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6: 2872-2833 (1986); al., J. Virology 57: 267-274 (1986); Davidson et al., J. Virology 61: 1226-1239 (1987); Zhang, “Recombinant Adenovirus Generation and Identification by Liposome-mediated Transduction and PCR Analysis BioTechniques 15: 868-872 (1993)). The advantage of using these viruses as vectors is limited to the extent that they can spread to other cell types because they can replicate in the originally infected cell, but new infections Infectious virus particles cannot be formed. Recombinant adenovirus has been demonstrated to achieve highly efficient gene transfer after direct in vivo transport to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and many other tissue sites (Morsy). J. Clin. Invest. 92: 1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92: 381-387 (1993); Roessler, J. Clin. Invest. 92: 1085-1092 (1993); Mullier, Nature Genetics 4: 154-159 (1993); La Salle, Science 259: 988-990 (1993); Gomez-Fix, J. Biol. Chem. 267: 25129-25134 (1992); an Gene Therapy 4: 461-476 (1993); Zabner, Nature Genetics 6: 75-83 (1994); Guzman, Circulation Research 73: 1201-1207 (1993); Bout, Human Gene Therapy 94 (3): 3). Zabner, Cell 75: 207-216 (1993); Caillaud, Eur. J. Neuroscience 5: 1287-1291 (1993); and Ragot, J. Gen. Virology 74: 501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated eating and drinking (Chardonnet and Dales), similar to wild-type or replication-deficient adenoviruses. Virology 40: 462-477 (1970); Brown and Burlingham, J. Virology 12: 386-396 (1973); Svensson and Persson, J. Virology 55: 442-449 (1985); 51: 650-655 (1984); Seth, et al., Mol. Cell. Biol.4: 1528-1533 (1984); rology65:. 6061~6070 (1991); Wickham et al, Cell73: 309~319 (1993)).

  Preferred viral vectors are adenovirus-based vectors with the E1 gene removed, and these virons are generated in cell lines such as the human 293 cell line. In another preferred embodiment, both the E1 and E3 genes are removed from the adenovirus genome.

  Another type of viral vector is based on adeno-associated virus (AAV). This defective parvovirus is a suitable vector because it can infect many cell types and is non-pathogenic to humans. It is known that AAV species vectors can transport about 4-5 kb and wild type AAV is stably inserted into chromosome 19. Vectors containing this site specific integration property are preferred. A particularly preferred embodiment of this type of vector is P4 made by Avigen, San Francisco, CA and containing a marker gene such as the herpes simplex virus thymidine kinase gene HSV-tk and / or the gene encoding the green fluorescent protein GFP. .1C vector.

Genes inserted into viruses and retroviruses typically include promoters and / or enhancers that help control the expression of the desired gene product. A promoter is generally a DNA sequence that functions when in a fixed position relative to the transcription start position. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and can contain upstream elements and reaction elements.
3. Viral promoters and enhancers Suitable promoters that control transcription from vectors in mammalian host cells include various sources such as polyoma, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis-B virus, and most preferably cytomegalo. It can be obtained from the genome of a virus, such as a virus, or from a heterologous mammalian promoter, such as the β-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, PJ et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species are also useful herein.

  Enhancers generally refer to functional DNA sequences that are not fixed in distance from the transcription start site and are 5 ′ to the transcription unit (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981). Or 3 ′ (Lusky, ML, et al., Mol. Cell Bio. 3: 1108 (1983)) In addition, enhancers are within introns (Banerji, JL et al., Cell 33: 729 (1983)) and within the coding sequence itself (Osborne, TF, et al., Mol. Cell Bio. 4: 1293 (1984)). Functions in cis at 10-300 bp Enhancer functions to increase transcription from nearby promoters Enhancers also often contain response elements that mediate the regulation of transcription, promoters can also contain reaction elements that mediate the regulation of transcription, and enhancers determine the regulation of gene expression. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, insulin) but usually use enhancers from eukaryotic viruses, a preferred example being the late origin of replication. SV40 enhancer (100 to 270 bp), cytomegalovirus early promoter enhancer late origin polyoma enhancer, and adenovirus enhancer.

  Promoters and / or enhancers can be specifically activated by light or specific chemical events that trigger function. The system can be regulated by reagents such as tetracycline and dexamethasone. There are also methods to improve viral vector gene expression by irradiation with gamma radiation or alkylating chemotherapy drugs.

  The promoter and / or enhancer region preferably serves as a constitutive promoter and / or enhancer in order to maximize expression of the region of the transcription unit that is transcribed. It is also preferred that the promoter and / or enhancer region be operable in all eukaryotic cell types. A suitable promoter of this type is the CMV promoter (650 bases). Other suitable promoters are the SV40 promoter, cytomegalovirus (full length promoter), and retroviral vector LTF.

  It has been demonstrated that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acidic protein (GFAP) promoter has been used to selectively express genes in cells originating from glia.

Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells) also contain sequences necessary for termination of transcription that may affect mRNA expression. Can do. These regions are transcribed as polyadenylation segments of the untranslated region of the mRNA encoding tissue factor protein. The 3 ′ untranslated region can also contain a transcription termination site. The transcription unit preferably includes a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcription unit will be processed and transported like mRNA. The identification and use of polyadenylation signals within expression constructs is well established. Homologous polyadenylation signals are preferably used in transgene constructs. In a preferred embodiment of the transcription unit, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcription unit be only another standard sequence or in combination with the above sequence to improve expression from the construct or stability of the construct.
4). Marker viral vectors can include a nucleic acid sequence that encodes a marker product. This marker product is used to determine if the gene has been carried into the cell and if it has been expressed once delivered. A preferred marker gene is the E. coli lacZ gene which encodes β-galactosidase and green fluorescent protein. In some embodiments, the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. If the selectable marker is successfully transferred to a mammalian host cell, the transformed mammalian host cell can survive when placed under selective pressure. There are two widely used separate category selection forms. The first category is based on cellular metabolism and the use of mutant cell lines that lack the ability to grow regardless of the supplemented medium. Two examples are CHO DHFR-cells and mouse LTK-cells. These cells lack the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. Since these cells lack certain genes required for the complete nucleotide synthesis pathway, they are not viable unless the missing nucleotides are provided in a supplemental medium. An alternative to supplemental media is to change growth requirements by introducing intact DHFR or TK genes into cells lacking each gene. Individual cells that have not been converted with the DHFR or TK gene cannot survive in non-supplemented media.

The second category refers to selection schemes used with any cell type and is the dominant selection that does not require the use of mutant cell lines. These schemes typically use agents that inhibit the growth of host cells. Those cells with the new gene express a protein conferring drug resistance, leaving the selection. Examples of such dominant selections are the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, RC and Berg, P. Science 209: 1422 (1980)) or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). Three examples employ bacterial genes under eukaryotic control to confer resistance to the appropriate drugs G418 or neomycin (Geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and plamycin.
S. Kits The materials described above and other materials can be packaged together in any suitable combination as a kit effective to perform the disclosed method or to assist in performing the disclosed method. It is useful if the kit components within a given kit are designed and adapted to be used together in the disclosed manner. For example, a kit for analyte analysis is disclosed that comprises a set of reporter signals and one or more indicator signals.
T. T. et al. Mixtures Disclosed are mixtures formed by performing or preparing to perform the disclosed method. For example, a mixture comprising a multidimensional signal, a reporter signal, an indicator signal, or a combination is disclosed.

Whenever the method involves mixing or contacting components, components or reagents, performing the method results in a number of different mixtures. For example, if the method has three mixing steps, after performing each of these steps, a unique mixture is formed if the steps are performed separately. Furthermore, regardless of how the steps are performed, the mixture is formed at the end of all steps. The present disclosure contemplates those mixtures obtained by performing the disclosed methods and mixtures of, for example, the disclosed reagents, components, or components disclosed herein.
U. Systems Disclosed are systems that are effective to perform or assist in performing the disclosed methods. Systems generally comprise structures, machines, devices, etc., and combinations of products such as components, compounds, materials, and the like. Combinations disclosed or obvious from the disclosure are also contemplated. For example, a system comprising a mass spectrometer having means for analyzing a pattern and selecting a portion of an analysis sample or further analysis is disclosed and contemplated.
V. Data Structure and Computer Control A data structure used in the disclosed method, generated by the disclosed method, or generated from the disclosed method is disclosed. A data structure is generally any form of data, information, and / or object that is collected, organized, stored, and / or embodied in a composition or medium. A protein signature stored in electronic form, such as in a RAM or storage disk, is a data structure.

The disclosed method, or a portion thereof, or preparation for it, can be controlled, managed, or otherwise assisted by computer control. The computer control can be accomplished by a computer-controlled process or method, can use and / or generate data structures, and can use computer programs. The above computer control, computer controlled process, data structure, and computer program are intended and should be understood as being disclosed herein.
Illustrative The disclosed method can be better understood with the following illustrative examples, including examples of the disclosed method. The illustration is not intended to limit the scope of the method in any way.
A. Example 1: A set of isobaric reporter and indicator signals; heavy isotopes This illustration shows the same mass of fragmenting specific peptide bonds and using heavy isotopes to differentially distribute mass with different reporter signals A peptide reporter signal is utilized. For example, ion traps have demonstrated that peptides containing arginine are preferentially fragmented at the C-terminus of aspartic acid or glutamic acid residues, and proline containing peptides are fragmented at the N-terminus of proline residues. (Qin and Chait, Int. J. Mass Spectrom. (Netherlands), 190-191: 313-20 (1999)). DP (aspartic acid (D) and proline (P)) amino acid sequences can be used with the disclosed reporter signal, resulting in collision-induced fragmentation at the cleavage bond between aspartic acid and proline.

A single charge ion of the typical peptide AGSLDPAGSLR (SEQ ID NO: 2) cleaves between “D” and “P” in the collision cell of the mass spectrometer. Using naturally abundant isotopes, the single charged parent ion has an average nominal (m / z) = 1043 amu, and the resulting daughter ion AGSLD ⁺ (SEQ ID NO: 2 amino acids 1-5) ) And PAGSLR ⁺ (amino acids 6-11 of SEQ ID NO: 2) have nominal (m / z) 461 amu and 600 amu, respectively. In effect, fragmentation usually results in one dominant daughter ion, in this case PAGSLR ⁺ (amino acids 6-11 of SEQ ID NO: 2). For this illustration, consider only one charged daughter from a single charged parent population. Without loss of generality or applicability, the branching ratio to these daughter ion channels can be other than 100% to PAGSLR ⁺ (amino acids 6-11 of SEQ ID NO: 2) daughter fragments. Please be careful.

Standard synthetic methods can be utilized to construct the peptides. In this reporter molecule illustration, consider isotope-labeled amino acids (eg, A vs. A ^* , where A has CH ₃ and A ^* has a CD ₃ side chain). For synthetic peptides having the nominal (m / z) shown in parentheses, AGSLDPAGSLR (1043), A ^* GSDLDPAGSLR (1046), AGSLDPA ^* GSLR (1046), A ^* GSLDPA ^* GSLR (1049) (SEQ ID NO: 2) There are four possibilities. In this example, two single-labeled peptides A ^* GSDLDPAGSLR, AGSLDPA with a general described mass to charge ratio of 1046 as the reporter signal and unlabeled peptide AGSLDPAGSLR (SEQ ID NO: 2), and an indicated mass to charge ratio of 1043 as the indicator signal ^* Consider GSLR (SEQ ID NO: 2).

  As a simple demonstration of a preferred mode of the disclosed method, consider a solution containing three synthetic peptides. This solution is collected after any number of biological experiments below and generally contains many additional components for processing.

A solution containing AGSLDPAGSLR, A ^* GSDLDPAGSLR, and AGSLDPA ^* GSLR (SEQ ID NO: 2) is mixed with an appropriate matrix solution to perform analysis by mass spectrometry. Suitable substrates such as sinapinic acid, 4-hydroxy-α-cyanocinnamic acid, or 2,5-dihydroxybenzoic acid are known in the art.

The resulting solution can be spotted on the MALDI target and crystallized. The target is inserted into a tandem mass spectrometer source of a quadrupole time-of-flight type (eg, Applied Biosystems QSTAR or Waters QtoF). Many ions are introduced into the first quadrupole Q0 using a laser that strikes the sample spot on the MALDI target. Among the species introduced into Q0, as is known in the art, most are single-charged species (AGSLDPAGSLR ⁺ , A ^* GSDLDPAGSLR ^{+ <“1”>} , AGSLDPA ^* GSLR ⁺ ; SEQ ID NO: 2), Various fragmented ions, neutral matrices, matrix ions, and multimers. Neutral particles disappear from Q0 without being guided into the second quadrupole Q1.

Ions introduced into Q0 operate in the DC field only (acting as an ion pipe rather than a mass / charge filter), are directed to a higher vacuum region including Q1, and are detected by a time-of-flight analyzer. The resulting spectrum (MS spectrum) is analyzed for each doublet peak separated by m / z = 3. Based on the identification of the doublet peak, the quadrupole Q1 is set to pass ions having a higher mass-to-charge doublet through the third quadrupole Q2 (A ^* GSDLDPAGSLR and AGSLDPA ^* GSLR (SEQ ID NO: Recall that 2) have the same mass-to-charge, “isobaric” in mass spectrometry terminology). Ions having a mass to charge ratio different from 1046 follow a trajectory that does not exit Q1 on the Q1-Q2 axis and are effectively truncated. This greatly increases the signal-to-noise of the system, an improvement of about 100-1000 times over this system without mass filtering.

Collision cells surrounding Q2 are filled with a chemically inert gas at the appropriate pressure to cause preferential cleavage of the peptide ion, usually several millitorr of argon or nitrogen DP cleavage bonds. As mentioned above, fragmentation of single charged parent ions is expected to result mainly in one daughter ion. In this case, each isobaric parent (SEQ ID NO: 2) results in a unique daughter (SEQ ID NO: 2 amino acids 1-5 and 6-11) that is correlated.
A ^* GSDLDPAGSLR ⁺ → A ^* GSLD + PAGSLR ⁺ (m / z 600)
AGSLDPA ^* GSLR ⁺ → AGSLD + PA ^* GSLR ⁺ (m / z 603)
Since the resolution of the mass spectrometer described herein is about 5000 to 10,000, a difference of 3 amu is easily obtained at these (m / z).

  Ions leaving Q2 enter the time-of-flight (TOF) part of the instrument. A transient electric field gradient is applied and the positively charged ions are accelerated towards the reflectron and finally to the detector. All ions are accelerated through the same electric field gradient (as is known in the art, the reflectron cancels out small disturbances in this determination), so the same kinetic energy is applied to all ions. Since the kinetic energy is the same for all ions and the masses of the ions are different, the time it takes for the ions to reach the detector is different. Heavy ions arrive later than light ions.

  The resulting mass spectrum (MS / MS spectrum) reflects the relative amount of two analytes (eg, peptides) in the initial sample.

The advantage of discriminating a given pattern (doublet peak separated by m / z = 3) and subsequent passage of a higher m / z peak in the doublet is in an assay that contains more multivariate signals of various m / z. It is more obvious. In such a case, the MS spectrum is analyzed for each doublet, and only the peaks included in the predetermined pattern are passed for recovery of the MS / MS spectrum. This scheme can be extended to more analytes (eg, peptides). The most basic extension for a panel of isobaric detectors based on the above peptides that utilizes the X / X ^* difference is shown in Table 2. An asterisk represents a heavy isotope-labeled amino acid. This set assumes that the change from unlabeled mass to labeled mass {(m / z) x ^* -(m / z) x} for each residue is the same. In the general case where {(m / z) x ^* -(m / z) x} is not the same for all residues, there are more given peptides that can be resolved by a mass spectrometer. The parent molecule is SEQ ID NO: 2, and the primary daughter is amino acids 6-11 of SEQ ID NO: 2.

特定同位体標識アミノ酸の合成は、パネルサイズの急速な増大を促進する。たとえば、ＣＨ_３、ＣＨ_２Ｄ、ＣＨＤ_２、ＣＤ_３側鎖と独自のアラニンの合成は、小さなペプチドで相当なパネルサイズをもたらすのに使用することができる。

The synthesis of specific isotope-labeled amino acids facilitates a rapid increase in panel size. For example, the synthesis of CH ₃ , CH ₂ D, CHD ₂ , CD ₃ side chains and a unique alanine can be used to yield considerable panel sizes with small peptides.

The disclosed method of this mode is that all detected ions originate from a very similar chemical environment (only differ in the position of a few neutrons) and are processed in the MALDI source and in the collision cell Sometimes it has the desired property of taking the same behavior (all for substantial purposes). Of particular note is the case where one of the isobaric reporter signal molecules is added as a metric for the isobaric detector molecule used for the assay. The metric of the entire set of detector molecules used in the assay is simple and quantitative. If the molecules are nearly identical except for isotopic enrichment, all isobaric nuclei behave identically throughout the process.
B. Example 2: Multi-dimensional signal of two isobaric sets; cleaved bond This example utilizes a peptide reporter signal with the same mass where the bond is cleaved at specific peptide bonds located at different positions in different reporter signals . As described above, DP having an amino acid sequence cleaves between aspartic acid and proline in collision cells. The multiple sets of peptides effective in the disclosed method may be:
Isobaric set 1:
Peptide C: YFMSGSGPGGR (SEQ ID NO: 13)
Peptide D: YFMTSGDPCGGR (SEQ ID NO: 14)
Peptide E: YFMTSDPCGCGGR (SEQ ID NO: 15)
Peptide F: YFMTDPSGCGGR (SEQ ID NO: 16)
Peptide G: YFMDPTSGCGGR (SEQ ID NO: 17)
Isobaric set 2:
Peptide H: YFMTSGCDPGAR (SEQ ID NO: 18)
Peptide I: YFMTSGDPCGAR (SEQ ID NO: 19)
Peptide J: YFMTSDPCGCGAR (SEQ ID NO: 20)
Peptide K: YFMTDPSGCGAR (SEQ ID NO: 21)
Peptide L: YFMDPTSGCGAR (SEQ ID NO: 22)
The two sets of peptides differ at the position of the DP dipeptide and at the amino acid at position 11 (glycine or alanine). The isobaric set 1 peptide differs from the isobaric set 2 peptide by 14 amu in mass (based on the mass difference between glycine and alanine).

  For simplicity, consider solutions containing these synthetic peptides. This solution can be recovered after any number of biological experiments and generally includes many additional components for processing. A solution containing C, D, E, F, G, H, I, J, K, and L is mixed with an appropriate matrix solution to perform analysis by mass spectrometry. Suitable matrices such as sinapinic acid, 4-hydroxy-α-cyanocinnamic acid, or 2,5-dihydroxybenzoic acid are known in the art.

  The resulting solution can be spotted on the MALDI target and crystallized.

  The target is inserted into the source of a tandem mass spectrometer of a quadrupole time-of-flight type (eg, Applied Biosystems QSTAR or Waters QtoF).

Many ions are introduced into the first quadrupole Q0 using a laser that strikes a spot on the MALDI target. Species introduced into Q0 are C ⁺ , D ⁺ , E ⁺ , F ⁺ , G ⁺ , H ⁺ , I ⁺ , J ⁺ , K ⁺ , L ⁺ , various fragmented ions as known in the art. , Matrix ions, and multimers. Neutral particles disappear from Q0 without being guided into Q1.

  Ions introduced into Q0 operate in the DC field only (acting as an ion pipe rather than a mass / charge filter), are directed to a higher vacuum region including Q1, and are detected by a time-of-flight analyzer. The resulting spectrum (MS spectrum) is analyzed for each doublet peak separated by m / z = 14. Based on the identification of the doublet peak, the quadrupole Q1 has a lower mass-to-charge doublet ((m / z) C, (m / z) D, (m / z) E, (m / z) F, (m / Z) G. They have the same molecular weight and are “isobaric”) and higher mass-to-charge doublets ((m / z) H, (m / z) I, (m / z ) J, (m / z) K, (m / z) L. They have the same molecular weight and are “isobaric”) set to individually pass through the third quadrupole Q2. Is done. (M / z) C, (m / z) D, (m / z) E, (m / z) F, (m / z) G, (m / z) H, (m / z) I, ( Ions having a mass-to-charge ratio different from (m / z) J, (m / z) K, and (m / z) L follow a trajectory that does not exit Q1 on the Q1-Q2 axis and are effectively truncated. This greatly increases the signal-to-noise of the system, an improvement of about 100-1000 times over this system without mass filtering.

The impinging cells surrounding Q2 are filled with a chemically inert gas at the appropriate pressure to cause cleavage of the DP bond, usually a few millitorr of argon or nitrogen. Considering only the ions with low mass-to-charge doublets, fragmentation at DP bonds, total charge retention by C-terminal fragments, and Q2 operation in RF-only mode, it appears from Q2 to the TOF part. There are five possible ions.
C1 ⁺ : PGGR ⁺ (SEQ ID NO: 13 amino acids 9 to 12)
D1 ⁺ : PCGGR ⁺ (SEQ ID NO: 14 amino acids 8 to 12)
E1 ⁺ : PGCGGR ⁺ (SEQ ID NO: 15 amino acids 7 to 12)
F1 ⁺ : PSGCCGGR ⁺ (SEQ ID NO: 16 amino acids 6 to 12)
G1 ⁺ : PTSCGCGGR ⁺ (SEQ ID NO: 17 amino acids 5 to 12)
A similar series of fragmented ions arises from Q2 analysis of ions with high mass to charge ratio doublets.

  Ions leaving Q2 enter the time-of-flight (TOF) part of the instrument. A transient electric field gradient is applied and the positively charged ions are accelerated towards the reflectron and finally to the detector. All ions are accelerated through the same electric field gradient (as is known in the art, the reflectron cancels out small disturbances in this decision), so the same kinetic energy is applied. Since the kinetic energy is the same for all ions and the masses of the ions are different, the time it takes for the ions to reach the detector is different. Heavy ions arrive later than light ions.

  The resulting mass spectrum (MS / MS spectrum) represents the relative amount of analyte (eg, peptide) in the initial sample.

  The advantage of discriminating a given pattern (doublet peak separated by m / z = 14) and subsequent passage of the peak in the doublet is more obvious in assays that contain more multivariate signals of various m / z. In such a case, the MS spectrum is analyzed for each doublet, and only the peaks included in the predetermined pattern are passed for recovery of the MS / MS spectrum.

A reference having the same mass as the analyte can be added to help obtain quantitative results. To derive quantitative results, the relative efficiency of the molecule under consideration should be determined for use in a simple process, calibration.
EXAMPLE This example provides an example of the disclosed method that includes labeling proteins with multidimensional signals and pattern recognition in the MS dimension for MS / MS data collection and analysis.

  Consider the two-sample assay shown in FIG. In this assay, bovine serum albumin (BSA) was selected as a typical protein. A typical BSA sample was split into two parts (composing two samples) and reacted with multiple sets of multidimensional signals (Table 3).

表３．システイン側鎖を標識付けするための多次元シグナル（標識）の選択された特性。Ｒｘは、アルキル化による共有結合を生成するスルフヒドリル反応性基（短リンカーを含む）を表す。ｇはグリシン残留物を表す。Ｇは、ｇに対して^１３Ｃ（２位置）と^１５Ｎ（１位置）で濃縮されたグリシン残留物を表す。標識セット１のメンバーは、６つの重グリシン残留物を組み込むため、標識セット２のメンバーより名目上１８ダルトン重いことに注意されたい。 Two sets of multi-dimensional labels were used (label set 1 and label set 2; see Table 3). The members of a given set are isobaric (all members of marker set 1 are isobaric to each other and all members of marker set 2 are isobaric to each other). That is, within the set, the label is isobaric. The set can be referred to as an isobaric set. The members of marker set 1 are not isobaric with the members of marker set 2. That is, marker set 1 and marker set 2 are not isobaric to each other. Table 3 shows the characteristics of the multidimensional signal.

Table 3. Selected properties of multidimensional signals (labels) for labeling cysteine side chains. Rx represents a sulfhydryl reactive group (including a short linker) that forms a covalent bond by alkylation. g represents glycine residue. G represents the glycine residue concentrated at ¹³ C (2 position) and ¹⁵ N (1 position) relative to g. Note that the tag set 1 members are nominally 18 dalton heavier than the tag set 2 members because they incorporate six heavy glycine residues.

  Bovine serum albumin (BSA, Sigma Cat # A7030) is dissolved in denaturing buffer (50 mM ammonium bicarbonate, pH 8.5, 6 M Urea, 0.5 mM Tris (2-carboxyl, phosphine hydrochloride, ie TCEP). The iPROT peptide label was synthesized and purified by American Peptido Co. Each label was dissolved in DMSO to a concentration of 10 mg / ml. Nominal isobaric cocktails were formulated by combining the same amount of isobaric sets of labels, one with 7 “heavy” labels (label set 1, Table 3) and 5 “light” labels. Two cocktails were made with (label set 2, Table 3) After denaturation, BS A was labeled by mixing 6 μg of label (either “heavy” or “light” cocktail) per μg of BSA and incubating in the dark at room temperature (24-25 ° C.) for 2 hours. The iPROT concentration for each labeling reaction was 3.6 mM After labeling, β-mercaptoethanol was added to a final concentration of 80 mM to eliminate excess label.

  A mixture of non-isobaric sets of labeled BSA was made by mixing equal amounts of “heavy” and “light” labeling reactions (see FIG. 1). The labeling solution mixture was then dialyzed against 0.1 M ammonium bicarbonate. Labeled BSA was digested with trypsin immobilized on agarose beads (PIERCE CAT # 20230). Initially, the beads were washed thoroughly in 0.1M ammonium bicarbonate and made as a 50% slurry. An amount of this slurry was mixed with an amount of dialyzed BSA solution and incubated overnight (˜16 hours) with stirring at 37 ° C. The supernatant was collected containing iPROT-labeled tryptic peptide.

  The resulting mixture was analyzed by LC / MS and LC / MS / MS. Sample peptides (representing trypsin fragments of BSA labeled with a multidimensional signal) were separated by reverse phase high performance liquid chromatography according to hydrophobicity, as is known in the art. Data were collected using an Agilent 1100LC connected to a Thermo Electron Corporation LTQ, or an Applied Biosystems / MDS Sciex QSTAR Pulsar with o-MALDI source. The resulting fragments were analyzed by MALDI tandem mass spectrometry and ESI tandem mass spectrometry. A typical spectrum of the LC process is shown in FIGS. 2A and 2B, which are graphs of mass spectrometry spectra of bovine serum albumin fragments labeled with multidimensional signals. FIG. 2A targets 1200 to 2500 m / z. FIG. 2B targets 500 to 1200 m / z. These spectra represent examples of indicator level analysis in the disclosed method in which a predetermined pattern is identified, MALDI data (FIG. 2A) is dominated by a single charge species (ie z = 1) and ESI (FIG. 2B) Is dominated by multi-charged species (z = 2, 3).

  The pattern of pairs of ions is highly recognizable and represents several ionic species. FIG. 2A targets 1200 to 2500 m / z. FIG. 2B targets 500 to 1200 m / z. These spectra represent examples of indicator level analysis in the disclosed method in which a predetermined pattern is identified. FIG. 2A is from a MALDI QSTAR device. Doublets spaced 18 daltons correspond to the mass difference between the members of Label Set 1 (heavy) and Label Set 2 (light) shown in Table 3. Pairs close to m / z = 1360 are spaced every 36 daltons corresponding to peptides with two cysteines, and thus two multidimensional signals. The presence of two multidimensional signals doubles the mass difference between the fragments labeled with the members of Label Set 1 and Label Set 2. FIG. 2B is from ESI LTQ FTMS. Doublets spaced 18 daltons correspond to the mass difference between the members of Label Set 1 (heavy) and Label Set 2 (light) shown in Table 3. These doublets (spaced in multiples of 18 Daltons) represent the predetermined pattern expected from the use of multi-dimensional labels in marker set 1 and marker set 2. These individual ionic species can be derived by performing MS / MS experiments.

  FIGS. 3A and 3B showing typical MS / MS spectra from an ESI LTQ FTMS instrument are graphs of mass spectrometry spectra of bovine serum albumin fragments displayed with multidimensional signals. These spectra show examples of reporter level analysis in the disclosed method, where the sample portion identified by the predetermined pattern undergoes further analysis (in this case MS / MS). FIG. 3A is an MS / MS spectrum having a peak at 898.44 m / z shown in FIG. 2B (the lighter peak of the doublet). This peak represents the sample portion analyzed in FIG. 2B identified for further analysis shown in FIG. 3 based on a predetermined pattern (peak doublets spaced in multiples of 18 Daltons). This peak represents a protein fragment labeled with a multidimensional signal from label set 2 (lighter set; see Table 3). The multidimensional signal is fragmented with DP residues in the signal, producing a pair of characteristic mass fragments. The two sets of five peaks in FIG. 3A show fragment pairs resulting from fragmentation of the multidimensional signal (one peak in one set is paired with one peak in another set). A set of five peaks is separated about every 60 Daltons. The spectra in FIGS. 3A and 3B are graphs of mass spectrometry spectra of bovine serum albumin fragments labeled with multidimensional signals. These spectra show examples of reporter level analysis in the disclosed method, where the sample portion identified by the predetermined pattern undergoes further analysis (in this case MS / MS). FIG. 3A is an MS / MS spectrum having a peak at 898.44 m / z shown in FIG. 2B (the lighter peak of the doublet). This peak represents the sample portion analyzed in FIG. 2B identified for further analysis shown in FIG. 3A based on a predetermined pattern (peak doublets spaced in multiples of 18 Daltons). This peak represents a protein fragment labeled with a multidimensional signal from label set 2 (lighter set; see Table 3). The multidimensional signal is fragmented with DP residues in the signal, producing a pair of characteristic mass fragments. The two sets of five peaks in FIG. 3A show fragment pairs resulting from fragmentation of the multidimensional signal (one peak in one set is paired with one peak in another set). A set of five peaks separated about every 60 Daltons, responsible for the selection of ions with a double charge state close to m / z = 900, are the bovine serum albumin fragments labeled with multidimensional signals. FIG. FIG. 2A targets 1200 to 2500 m / z. FIG. 2B targets 500 to 1200 m / z. These spectra show examples of indicator level analysis in the disclosed method where a predetermined pattern is identified. After one peak of the BC doublet is analyzed in FIG. 3A and the other is analyzed in FIG. 3B, two single charge fragments result from collision-induced fragmentation (one group is about m / z = 460). And the other group is centered around m / z = 1350). FIG. 3A is an MS / MS spectrum having a peak at 898.44 m / z shown in FIG. 2B (the lighter peak of the doublet). This peak represents the sample portion analyzed in FIG. 2B identified for further analysis shown in FIG. 3A based on a predetermined pattern (peak doublets spaced in multiples of 18 Daltons). This peak represents a protein fragment labeled with a multidimensional signal from label set 2 (lighter set; see Table 3). The multidimensional signal is fragmented with DP residues in the signal, producing a pair of characteristic mass fragments. The two sets of five peaks in FIG. 3A show fragment pairs resulting from fragmentation of the multidimensional signal (one peak in one set is paired with one peak in another set). A set of five peaks is separated about every 60 Daltons.

  FIG. 3B is an MS / MS spectrum having a peak at 907.45 m / z shown in FIG. 2B (the peak of the heavier doublet). This peak represents the sample portion analyzed in FIG. 2B identified for further analysis shown in FIG. 3B based on a predetermined pattern (peak doublets spaced in multiples of 18 Daltons). This peak represents a protein fragment that is labeled with a multidimensional signal from label set 1 (heavy set; see Table 3). The multidimensional signal is fragmented with DP residues in the signal, producing a pair of characteristic mass fragments. The two sets of seven peaks in FIG. 3B (closer intervals in the graph) show pairs of fragments resulting from fragmentation of multidimensional signals (one peak in one set is in another set) Pair with one peak). A set of seven peaks are separated approximately every 3 daltons (not well analyzed at the resolution of the graph).

  It is understood that the disclosed methods and configurations are not limited to the specific methodologies, protocols, and reagents described above, and can be varied. Also, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the invention, which is limited only by the appended claims. It is understood that it is limited.

As used in this specification and the appended claims, it should be understood that a singular expression includes the plural expression unless the context clearly indicates otherwise. Thus, for example, reference to “reporter signal” includes a plurality of reporter signals, and reference to “oligonucleotide” refers to one or more oligonucleotides and equivalents known to those of skill in the art.
“Any” or “optionally” means that an event, situation, or material described later may or may not exist, and that the explanation is an event, situation Or examples where material occurs or exists, events, situations, or examples where material does not occur or does not exist.

  Ranges can be expressed herein as “approximately” from one particular value and / or to “approximately” another particular value. Where the above range is indicated, it is intended and considered that the range from one particular value and / or to another particular value is explicitly disclosed, unless the context specifically indicates otherwise. The Similarly, when a value is indicated in approximate terms using the antecedent “about,” the particular value is intended to be other specifically intended to be considered disclosed unless the context clearly indicates otherwise. It is understood that this embodiment forms an embodiment. Each endpoint of a range is understood to be valid relative to and independent of other endpoints unless the context clearly indicates otherwise. Finally, all values within a clearly disclosed range and sub-ranges of values within that range are also specifically intended and should be considered disclosed unless the context clearly indicates otherwise. It should be understood that there is. The foregoing applies regardless of whether in particular cases, some or all of these embodiments are explicitly disclosed.

  Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the disclosed method and composition belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions, particularly useful methods, devices, and materials are described. The publications and materials cited herein are specifically incorporated herein by reference. No part of the specification should be construed as an admission that the invention is not entitled to antedate the above disclosure by virtue of prior invention. No reference is admitted to constitute prior art. The references to the references state what the authors claim, and the applicant has the right to challenge the accuracy and relevance of the cited documents. Many publications are mentioned in this specification, but that reference does not admit that any of the literature forms part of the general knowledge of the art.

  Throughout the specification and claims, variations such as “comprising” and “comprising”, “comprising” mean “including but not limited to”, eg, other additives, components It should be understood that it is not intended to exclude integers, or steps.

  Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

FIG. 1 is a diagram illustrating an example of using a multidimensional signal. The two samples are individually labeled with two sets of multidimensional signals (label set 1 and label set 2). The labeled sample is mixed and subjected to trypsin digestion (this cleaves the protein in the sample). The mixed and trypsinized sample is clarified by HPLC and subjected to two mass analyses. Example 1 is an example of an assay that follows the steps shown in FIG. Figures 2A and 2B are graphs of mass spectrometry spectra of bovine serum albumin fragments labeled with multidimensional signals. FIG. 2A targets 1200 to 2500 m / z. FIG. 2B targets 500 to 1200 m / z. These spectra show an example of indicator level analysis of the disclosed method that can identify a predetermined pattern. FIG. 2A is from a MALDI QSTAR device. Doublets spaced 18 daltons correspond to the mass difference between the members of Label Set 1 (heavy) and Label Set 2 (light) shown in Table 3. Pairs close to m / z = 1360 correspond to peptides with 2 cysteines and thus 2 multidimensional signals, spaced 36 daltons. The presence of two multidimensional signals doubles the mass difference between the fragments labeled with the members of Label Set 1 and Label Set 2. FIG. 2B is from ESI LTQ FTMS. Doublets spaced 18 daltons correspond to the mass difference between the members of Label Set 1 (heavy) and Label Set 2 (light) shown in Table 3. These doublets (spaced in multiples of 18 daltons) represent the predetermined pattern expected from the use of multidimensional labels in label set 1 and label set 2. Example 1 describes the generation of the graphs of FIGS. 2A and 2B. Figures 2A and 2B are graphs of mass spectrometry spectra of bovine serum albumin fragments labeled with multidimensional signals. FIG. 2A targets 1200 to 2500 m / z. FIG. 2B targets 500 to 1200 m / z. These spectra show an example of indicator level analysis of the disclosed method that can identify a predetermined pattern. FIG. 2A is from a MALDI QSTAR device. Doublets spaced 18 daltons correspond to the mass difference between the members of Label Set 1 (heavy) and Label Set 2 (light) shown in Table 3. Pairs close to m / z = 1360 correspond to peptides with 2 cysteines and thus 2 multidimensional signals, spaced 36 daltons. The presence of two multidimensional signals doubles the mass difference between the fragments labeled with the members of Label Set 1 and Label Set 2. FIG. 2B is from ESI LTQ FTMS. Doublets spaced 18 daltons correspond to the mass difference between the members of Label Set 1 (heavy) and Label Set 2 (light) shown in Table 3. These doublets (spaced in multiples of 18 daltons) represent the predetermined pattern expected from the use of multidimensional labels in label set 1 and label set 2. Example 1 describes the generation of the graphs of FIGS. 2A and 2B. Figures 3A and 3B are graphs of mass spectrometry spectra of bovine serum albumin fragments labeled with multidimensional signals. These spectra show examples of reporter level analysis in the disclosed method (in this case MS / MS) where the portion of the sample identified by the predetermined pattern undergoes further analysis. FIG. 3A is an MS / MS spectrum having a peak at 898.44 m / z shown in FIG. 2B (the lighter peak of the doublet). This peak represents the sample portion analyzed in FIG. 2B identified for further analysis shown in FIG. 3A based on a predetermined pattern (peak doublets spaced in multiples of 18 Daltons). This peak represents a protein fragment labeled with a multidimensional signal from label set 2 (lighter set; see Table 3). The multidimensional signal is fragmented with DP residues in the signal, producing a pair of characteristic mass fragments. The two sets of five peaks in FIG. 3A show fragment pairs resulting from fragmentation of the multidimensional signal (one peak in one set is paired with one peak in another set). A set of five peaks is separated about every 57 daltons.

FIG. 3B is an MS / MS spectrum having a peak at 907.45 m / z shown in FIG. 2B (the peak of the heavier doublet). This peak represents the sample portion analyzed in FIG. 2B identified for further analysis shown in FIG. 3B based on a predetermined pattern (peak doublets spaced in multiples of 18 Daltons). This peak represents a protein fragment that is labeled with a multidimensional signal from label set 1 (heavy set; see Table 3). The multidimensional signal is fragmented with DP residues in the signal, producing a pair of characteristic mass fragments. The two sets of seven peaks in FIG. 3B (closer intervals in the graph) show pairs of fragments resulting from fragmentation of multidimensional signals (one peak in one set is in another set) Pair with one peak). A set of seven peaks are separated approximately every 3 daltons (not well analyzed at the resolution of the graph). Example 1 describes the generation of the graphs of FIGS. 3A and 3B.
Figures 3A and 3B are graphs of mass spectrometry spectra of bovine serum albumin fragments labeled with multidimensional signals. These spectra show examples of reporter level analysis in the disclosed method (in this case MS / MS) where the portion of the sample identified by the predetermined pattern undergoes further analysis. FIG. 3A is an MS / MS spectrum having a peak at 898.44 m / z shown in FIG. 2B (the lighter peak of the doublet). This peak represents the sample portion analyzed in FIG. 2B identified for further analysis shown in FIG. 3A based on a predetermined pattern (peak doublets spaced in multiples of 18 Daltons). This peak represents a protein fragment labeled with a multidimensional signal from label set 2 (lighter set; see Table 3). The multidimensional signal is fragmented with DP residues in the signal, producing a pair of characteristic mass fragments. The two sets of five peaks in FIG. 3A show fragment pairs resulting from fragmentation of multidimensional signals (one peak in one set is paired with one peak in another set). A set of five peaks is separated about every 57 daltons.

FIG. 3B is an MS / MS spectrum having a peak at 907.45 m / z shown in FIG. 2B (the peak of the heavier doublet). This peak represents the sample portion analyzed in FIG. 2B identified for further analysis shown in FIG. 3B based on a predetermined pattern (peak doublets spaced in multiples of 18 Daltons). This peak represents a protein fragment that is labeled with a multidimensional signal from label set 1 (heavy set; see Table 3). The multidimensional signal is fragmented with DP residues in the signal, producing a pair of characteristic mass fragments. The two sets of seven peaks in FIG. 3B (closer intervals in the graph) show pairs of fragments resulting from fragmentation of multidimensional signals (one peak in one set is in another set) Pair with one peak). A set of seven peaks are separated approximately every 3 daltons (not well analyzed at the resolution of the graph). Example 1 describes the generation of the graphs of FIGS. 3A and 3B.
4A and 4B are diagrams illustrating example logic flows in the disclosed method. In FIG. 4A, mass spectrometry spectra are collected (first frame) and the spectra are analyzed to detect non-isobaric patterns (second frame). The first two panes correspond to indicator level analysis. The spectrum can be scanned for a predetermined pattern (first circle). If the predetermined pattern is not detected, the indicator level analysis is repeated for another sample or sample portion (loop from the first circle to the first frame). If a predetermined pattern is detected, the sample portion where the pattern was detected is sent for another level of analysis (first circle; down arrow). A tandem mass spectrometry spectrum is collected on the sample portion (third frame) and the spectrum is analyzed to obtain information about the sample. The third and fourth frames correspond to analysis of reporter signal levels. The entire analysis can be repeated on additional samples (loop from second circle to first frame).

In FIG. 4B, mass spectrometry spectra are collected (first frame). A tandem mass spectrometry spectrum is collected on the sample portion (second pane). The first two stages can be repeated on additional samples (loop from first circle to first frame). The spectrum is analyzed to detect non-isobaric patterns (third frame) and the spectrum is analyzed to obtain information about the sample (fourth frame). The first and third boxes correspond to indicator level analysis. The second and fourth frames correspond to analysis of reporter signal levels. FIG. 4B is an example separation of the data collection and data analysis portions of the level analysis in the disclosed method. FIG. 4 is an example of an analysis that can be included within and between the two mass analysis stages shown in FIG. Example 1 provides an example of the use of the logic flow shown in FIG.
4A and 4B are diagrams illustrating example logic flows in the disclosed method. In FIG. 4A, mass spectrometry spectra are collected (first frame) and the spectra are analyzed to detect non-isobaric patterns (second frame). The first two panes correspond to indicator level analysis. The spectrum can be scanned for a predetermined pattern (first circle). If the predetermined pattern is not detected, the indicator level analysis is repeated for another sample or sample portion (loop from the first circle to the first frame). If a predetermined pattern is detected, the sample portion where the pattern was detected is sent for another level of analysis (first circle; down arrow). A tandem mass spectrometry spectrum is collected on the sample portion (third frame) and the spectrum is analyzed to obtain information about the sample. The third and fourth frames correspond to analysis of reporter signal levels. The entire analysis can be repeated on additional samples (loop from second circle to first frame).

In FIG. 4B, mass spectrometry spectra are collected (first frame). A tandem mass spectrometry spectrum is collected on the sample portion (second pane). The first two stages can be repeated on additional samples (loop from first circle to first frame). The spectrum is analyzed to detect non-isobaric patterns (third frame) and the spectrum is analyzed to obtain information about the sample (fourth frame). The first and third panes correspond to indicator level analysis. The second and fourth frames correspond to analysis of reporter signal levels. FIG. 4B is an example separation of the data collection and data analysis portions of the level analysis in the disclosed method. FIG. 4 is an example of an analysis that can be included within and between the two mass analysis stages shown in FIG. Example 1 provides a usage example of the logic flow shown in FIG.
FIG. 5 is a diagram illustrating an example of using a multidimensional signal. FIG. 5 illustrates the method shown in FIG. 1 in which two different sample sets (control sample and test sample) are labeled with different members of two different sets of multidimensional signals (labeling set 1 and labeling set 2). An example is shown. In this example, five different test samples are each labeled with a different label set 2 member and seven different control samples are each labeled with a different label set 1 member. The marker set may be, for example, the marker set shown in Table 3. The correlation between the label set and the control and test samples is for clarity and does not indicate a limitation of the method. The labeled sample is mixed and subjected to trypsin digestion (this cleaves the protein in the sample). Mixed and trypsinized samples are clarified by HPLC and subjected to two mass analyses. In a preferred type of method, the label control and test sample are mixed throughout label set 1 and label set 2. 6A, 6B, and 6C are structural diagrams of iTRAQ multiple isobaric labeling chemistry. FIG. 6A shows a complete molecule consisting of a reporter group (based on N-methylpiperazine), a mass balance group (carbonyl), and a peptide reactive group (NHS ester). Since the mass of the reporter group is 114.1 to 117.1 m / z and the mass of the balance group is 28 to 31 daltons, the combined mass is constant for each of the four reagents (145.1 daltons). FIG. 6B shows the structure when the tag reacts with the peptide to form an amide linkage to the peptide amine (N-terminal or epsilon amino group of lysine). FIG. 6C shows isotope tagging used to arrive at four isobaric combinations with four different reporter group masses (left). A mixture of four identical peptides, each labeled with one member of the multiplex set, appears as a single undivided precursor ion in the MS (identical m / z; middle). After collision-induced dissociation (CID), the four reporter group ions appear as separate masses (114-117 daltons; right). 6A, 6B, and 6C are structural diagrams of iTRAQ multiple isobaric labeling chemistry. FIG. 6A shows a complete molecule consisting of a reporter group (based on N-methylpiperazine), a mass balance group (carbonyl), and a peptide reactive group (NHS ester). Since the mass of the reporter group is 114.1 to 117.1 m / z and the mass of the balance group is 28 to 31 daltons, the combined mass is constant for each of the four reagents (145.1 daltons). FIG. 6B shows the structure when the tag reacts with the peptide to form an amide linkage to the peptide amine (N-terminal or epsilon amino group of lysine). FIG. 6C shows isotope tagging used to arrive at four isobaric combinations with four different reporter group masses (left). A mixture of four identical peptides, each labeled with one member of the multiplex set, appears as a single undivided precursor ion in the MS (identical m / z; middle). After collision-induced dissociation (CID), the four reporter group ions appear as separate masses (114-117 daltons; right). 6A, 6B, and 6C are structural diagrams of iTRAQ multiple isobaric labeling chemistry. FIG. 6A shows a complete molecule consisting of a reporter group (based on N-methylpiperazine), a mass balance group (carbonyl), and a peptide reactive group (NHS ester). Since the mass of the reporter group is 114.1 to 117.1 m / z and the mass of the balance group is 28 to 31 daltons, the combined mass is constant for each of the four reagents (145.1 daltons). FIG. 6B shows the structure when the tag reacts with the peptide to form an amide linkage to the peptide amine (N-terminal or epsilon amino group of lysine). FIG. 6C shows isotope tagging used to arrive at four isobaric combinations with four different reporter group masses (left). A mixture of four identical peptides, each labeled with one member of the multiplex set, appears as a single undivided precursor ion in the MS (identical m / z; middle). After collision-induced dissociation (CID), the four reporter group ions appear as separate masses (114-117 daltons; right). FIG. 7 shows the peptide THPPARTEAK from a protein digestion mixture made by labeling four separate digests with each of the four isobaric reagents and combining the reaction mixture in a 1: 1: 1: 1 ratio. An example of an MS / MS spectrum is shown. The isotope distribution of the precursor ([M + H] +, 1352.84 m / z) is shown in i). The components within the spectrum frame shown in the center are shown at the bottom. ii) shows the low mass region indicating the signature ion used for quantification, iii) shows the isotope distribution of the b ₆ fragment, and iv) shows the isotope distribution of the Y ₇ fragment. Peptides are labeled with isobaric tags at both the N-terminal and C-terminal lysine side chains. Thus, all of the precursor ions and internal fragment ions (eg, type b- and type y-) contain all four members in the tag set, but remain isobaric. The example shown is a spectrum obtained from a single charge [M + H] + peptide using a 4700 MALDITF-TOF analyzer.

Claims (33)

A multidimensional signal group comprising a set of reporter signals and one or more indicator signals,
The set of reporter signals comprises a plurality of reporter signals;
The reporter signal has a common property, which allows the reporter signal to be distinguished or separated from molecules lacking the common property,
The reporter signal can be altered, each reporter signal in the modified form can be distinguished from any other modified form of the reporter signal,
At least one of the indicator signals does not have a common characteristic,
At least one of the set of reporter signals and indicator signals generates a predetermined pattern in a situation where the common property can distinguish or separate the reporter signal from molecules lacking the common property;
A multidimensional signal group characterized by that. A common property is the mass-to-charge ratio, the reporter signal is altered by changing the mass, and the altered form of the reporter signal is distinguishable via the mass-to-charge ratio difference of the altered form of the reporter signal, The set of claim 1, wherein the predetermined pattern is a mass-to-charge ratio pattern. A multidimensional signal group comprising two or more sets of reporter signals and one or more indicator signals,
Each set of reporter signals has multiple reporter signals, the reporter signals in each set have common characteristics, and the common characteristics can distinguish or separate the reporter signals in the set from molecules lacking common characteristics. , The reporter signal is changeable, each reporter signal in the modified form in each set is distinguishable from all other modified forms of the reporter signal in the set,
The common characteristics of the reporter signals in each set differ from the common characteristics of another set of reporter signals, at least one of the indicator signals does not have a common characteristic,
A group of multi-dimensional signals, characterized in that at least one of the set of reporter signals and indicator signals generates a predetermined pattern in a situation where the common property can distinguish or separate the reporter signal from molecules lacking the common property. A common property is the mass-to-charge ratio, the reporter signal is altered by changing the mass, and the altered form of the reporter signal is distinguishable via the mass-to-charge ratio difference of the altered form of the reporter signal, 4. The set of claim 3, wherein the predetermined pattern is a mass to charge ratio pattern. (A) separating a reporter signal and one or more indicator signals each having a common property from a molecule lacking the common property;
(B) identifying a predetermined pattern generated from at least one of a set of reporter signals and indicator signals;
(C) changing the reporter signal that generated the predetermined pattern;
(D) detecting and distinguishing from each other a modified form of the reporter signal;
A method comprising the steps of: A common property is the mass-to-charge ratio, the reporter signal is altered by changing the mass, and the altered form of the reporter signal is differentiated via the mass-to-charge ratio difference of the altered form of the reporter signal, with a predetermined pattern 6. The method of claim 5, wherein is a mass to charge ratio pattern. 6. The method of claim 5, wherein the reporter signal is associated with or bound to a specific binding molecule, and each reporter signal is associated with or bound to a different specific binding molecule. 6. The method of claim 5, wherein the reporter signal comprises a peptide, the peptide has the same mass to charge ratio, and the at least one indicator signal has a different mass to charge ratio than the reporter signal. Prior to step (a), the method further comprises associating a reporter signal and an indicator signal with one or more analytes, wherein each reporter signal and indicator signal is associated with or bound to a different specific binding molecule. 6. The method of claim 5, wherein the can specifically interact with different ones of the analytes, and the reporter signal and indicator signal are associated with the analytes through the interaction of the specific binding molecule and the analyte. Prior to step (a), further comprising associating a reporter signal with one or more analytes, wherein each reporter signal is associated with or bound to a different specific binding molecule, each specific binding molecule being different among the analytes. The at least one indicator signal is associated with or bound to a specific binding molecule having the same specificity as the specific binding molecule to which the reporter signal is associated or bound. 6. The method of claim 5, wherein the indicator signal is associated with the analyte through interaction of the specific binding molecule with the analyte. 6. The method of claim 5, wherein steps (a)-(dc) are repeated one or more times, each time using a different set of reporter signals. 6. The method of claim 5, wherein steps (b)-(d) are repeated one or more times, each time using a different set of reporter signals. 13. The method of claim 12, wherein prior to step (a), different sets of reporter signals are associated with different samples. The method of claim 12, wherein each of the plurality of sets of reporter signals comprises a single reporter signal. (A) separating two or more sets of reporter signals and one or more indicator signals from molecules lacking common characteristics of the two or more sets, wherein the reporter signals in each set have common characteristics The common characteristics of the reporter signals in each set are different from the common characteristics of another set of reporter signals;
(B) identifying a predetermined pattern generated by multiple sets of reporter signals and at least one indicator signal;
(C) changing the reporter signal that generated the predetermined pattern;
(D) detecting and distinguishing from each other a modified form of the reporter signal. A common property is the mass-to-charge ratio, the reporter signal is altered by changing its mass, and the altered form of the reporter signal is distinguished via the difference in mass-to-charge ratio of the altered form of the reporter signal The method of claim 15, wherein the pattern is a mass to charge ratio pattern. (A) a set of reporter molecules, each reporter molecule comprising a reporter signal and a decoding tag;
Reporter signals have common properties that allow them to distinguish or separate reporter signals from molecules that lack common properties,
The reporter signal can be changed, each reporter signal in the modified form is distinguishable from all other modified forms of the reporter signal,
Different reporter molecules each having a different decoding tag and a different reporter signal; and
(B) one or more indicator signals, wherein the at least one indicator signal has no common characteristics;
In situations where the common property allows the reporter signal to be distinguished or separated from molecules lacking the common property, the set of reporter signals and the at least one indicator signal generate an indicator signal, and
A set of encoding molecules, each encoding molecule having a specific binding molecule and an encoding tag, each specific binding molecule can interact clearly with a different analyte, and each encoding tag has a different decoding And a coding molecule that can clearly interact with the tag. (A) two or more sets of reporter molecules, wherein the reporter signals within each set have common characteristics;
The common property allows the reporter signal to be distinguished or separated from molecules lacking the common property, the common property of the reporter signal in each set is different from the common property of another set of reporter signals,
The reporter signal is changeable, and each reporter signal in the modified form in each set is distinguishable from all other modified forms of the reporter signal in the set; and
(B) one or more indicator signals, wherein the at least one indicator signal has no common characteristics;
In situations where the common property allows the reporter signal to be distinguished or separated from molecules lacking the common property, the set of reporter signals and the at least one indicator signal comprise an indicator signal that produces a predetermined pattern kit. Labeling the analyte in the first sample or the first set of samples with a set of multidimensional signals;
Labeling a second sample or an analyte in a second set of samples with a different set of multidimensional signals;
Mixing the first and second samples to form an analytical sample;
Multidimensional signal in an analytical sample—analyzing a labeled analyte to identify one or more predetermined patterns resulting from the multidimensional signal, wherein the identification of the one or more predetermined patterns is one or more portions of the analytical sample Identifying a step;
Analyzing the multidimensional signal in one or more of the one or more identification portions of the analysis sample to identify the multidimensional signal present in the identification portion of the analysis sample. Labeling a first sample or an analyte in the first set of samples with a first set of multidimensional signals;
Labeling a second sample or an analyte in a second set of samples with a second set of multi-dimensional signals, the first set of multi-dimensional signal members, a second set of multi-dimensions Having one or more common characteristics that can distinguish and / or separate a multidimensional signal in which members of the signal, or both, have common characteristics from another molecule lacking other common characteristics;
Mixing the first and second samples to form an analytical sample;
Multidimensional signal in an analytical sample—analyzing a labeled analyte to identify one or more predetermined patterns resulting from the multidimensional signal, wherein the identification of the one or more predetermined patterns is one or more portions of the analytical sample Identifying a step;
Analyzing a multidimensional signal in one or more of the one or more identification portions of the analysis sample to identify the multidimensional signal present in the identification portion of the analysis sample, wherein the one or more common characteristics are And a member of the set comprising: changing so that different types of different multidimensional signals in the changed form are distinguishable from each other. Labeling an analyte in the first sample or first set of samples with one or more isobaric multidimensional signals or one or more sets of isobaric multidimensional signals;
Labeling analytes in the second sample or second set of samples with one or more different MDSs or one or more different sets of multidimensional signals;
Mixing the first and second samples to form an analytical sample;
Multidimensional signal in an analytical sample—analyzing a labeled analyte to identify one or more predetermined patterns resulting from the multidimensional signal, wherein the identification of the one or more predetermined patterns is one or more portions of the analytical sample Identifying a step;
Analyzing a multidimensional signal in one or more of the one or more identification portions of the analysis sample to identify a multidimensional signal present in the identification portion of the analysis sample, the method comprising: Analysis of multi-dimensional signals in one or more discriminating parts can be performed by fragmenting the multi-dimensional signal in the discriminating part to create multi-dimensional signal fragments having different masses and detecting different multi-dimensional signal fragments based on mass-to-charge ratios. And the step achieved by the method. A multidimensional signal group comprising a set of reporter signals and one or more indicator signals,
The set of reporter signals comprises a plurality of reporter signals, the reporter signals have common characteristics,
The common property allows the reporter signal to be distinguished and / or separated from molecules lacking the common property,
The reporter signal can be changed, each reporter signal in the modified form is distinguishable from all other modified forms of the reporter signal,
A multidimensional group of signals characterized in that the reporter signal and the one or more indicator signals generate a predetermined pattern in a situation where the common property allows the reporter signal to be distinguished and / or separated from molecules lacking the common property. A set of labeled proteins, each labeled protein comprising a protein or peptide and a reporter signal or indicator signal added to the protein or peptide, the reporter signals having common characteristics, depending on the common characteristics, Distinguishing and / or separating labeled proteins comprising peptides from molecules lacking common properties,
The reporter signal can be modified, each reporter signal in the modified format can be distinguished from all other modified reporter signals, and changing the reporter signal can alter the labeled protein, Each labeled protein is distinguishable from all other modified forms of labeled protein,
A labeled protein characterized in that the reporter signal and the one or more indicator signals generate a predetermined pattern in a situation where the common property allows the labeled protein to be distinguished and / or separated from molecules lacking the common property. A set of reporter signal calibrators and one or more indicator signal calibrators, each reporter signal calibrator sharing a common characteristic with a target protein fragment within a set of target protein fragments, and having a common characteristic by a common characteristic Protein fragments and reporter signal calibrators can be distinguished and / or separated from molecules lacking common properties, target protein fragments and reporter signal calibrators that share common properties correspond to each other,
The target protein fragment can be altered, the altered form of the target protein fragment can be distinguished from other altered forms of the target protein fragment, the reporter signal calibrator can be altered, and each reporter in the altered form Signal calibrators are distinguishable from altered forms of target protein fragments that reporter signal calibrators share common properties,
In a situation where target protein fragments and reporter signal calibrators having common properties can be distinguished and / or separated from molecules lacking common properties, the reporter signal calibrator and at least one indicator signal calibrator can generate a predetermined pattern. Feature reporter signal calibrator and indicator signal calibrator. A set of nucleic acid molecules,
Each nucleic acid molecule comprises a reporter signal peptide or indicator signal peptide and a nucleotide segment that encodes the amino acid segment comprising the protein or peptide. Can be distinguished and / or separated from molecules lacking properties,
The reporter signal peptide can be altered, each reporter signal peptide in the altered form is distinguishable from another reporter signal peptide in the altered form,
In situations where the common property allows the reporter signal peptide to be distinguished and / or separated from molecules lacking the common property, the reporter signal peptide and one or more indicator signal peptides produce a predetermined pattern;
A nucleic acid molecule characterized by the above. A detector comprising a specific binding molecule, a carrier, and a group of blocks, the group of blocks comprising a block, the block comprising a set of reporter signals and one or more indicator signals . A detector comprising a specific binding molecule, a carrier, and a group of blocks, wherein the group of blocks comprises a block, and the block comprises two or more sets of reporter signals. (A) separating a set of reporter signals and one or more indicator signals from molecules lacking common properties, each reporter signal having common properties;
(B) identifying a predetermined pattern generated by the reporter signal and the one or more indicator signals;
(C) changing a reporter signal that generates a predetermined pattern; and (d) detecting and distinguishing from each other a changed form of the reporter signal. (A) separating a set of labeled proteins, each labeled protein comprising a protein or peptide and a reporter signal or indicator signal added to the protein or peptide, each reporter signal having common characteristics The ability to distinguish and / or separate labeled proteins comprising the same protein or peptide from molecules lacking common properties by common properties;
(B) identifying a predetermined pattern generated by the reporter signal and the one or more indicator signals;
(C) changing the labeled protein by changing the reporter signal that generates the predetermined pattern;
And (d) detecting and distinguishing the modified forms of labeled proteins from each other. (A) processing a protein sample to produce a protein fragment, wherein the protein fragment comprises a set of target protein fragments, the target protein fragment can be altered, and the altered form of the target protein fragment is Steps distinguishable from other modified forms of target protein fragments;
(B) mixing the target protein fragment with a set of reporter signal calibrators and one or more indicator signal calibrators, each target protein fragment sharing common characteristics with at least one reporter signal calibrator, Allows target protein fragments and reporter signal calibrators with common characteristics to be distinguished and / or separated from molecules lacking common characteristics, target protein fragments and reporter signal calibrators that share common characteristics correspond to each other, and reporter signal calibrators change Each reporter signal calibrator in a modified format that is possible is separated from a modified form of the target protein fragment that the reporter signal calibrator shares common characteristics. Possible and the step is,
(C) separating the target protein fragment and the reporter signal calibrator from another molecule based on the common properties of the target protein fragment and the reporter signal calibrator;
(D) identifying a predetermined pattern generated by the reporter signal calibrator and the one or more indicator signal calibrators;
(E) changing the target protein fragment and reporter signal calibrator that generated the predetermined pattern;
(F) detecting a modified form of the target protein fragment and a reporter signal calibrator, wherein the presence, absence, amount, or presence and amount of the modified form of the target protein fragment is determined by the modified form of the target Indicates the presence, absence, amount, or presence and amount of the target protein fragment in the protein sample from which the protein fragment is obtained, and the presence, absence, amount, or presence and amount of the target protein fragment in the protein sample indicates the protein signature of the protein sample Comprising the steps of:
A method of creating a protein signature, comprising: Detecting a target altered reporter signal peptide obtained from one or more expression samples, the one or more expression samples collectively comprising a set of nucleic acid molecules, each nucleic acid molecule comprising a reporter signal peptide or indicator Comprising a signal peptide and a nucleotide segment encoding an amino acid segment comprising the protein or peptide, wherein the reporter signal peptide has common properties, and the common property distinguishes the reporter signal peptide from molecules lacking common properties and / or A method of detecting expression comprising a step that can be separated, comprising:
The reporter signal peptide can be altered, each reporter signal peptide in the altered form can be distinguished from another reporter signal peptide in the altered form, and the target altered reporter signal peptide is one of the altered reporter signal peptides. One, detection of the target altered reporter signal peptide indicates expression of an amino acid segment comprising the reporter signal peptide from which the target altered reporter signal peptide is obtained;
A method characterized in that the reporter signal peptide and one or more indicator signal peptides generate a predetermined pattern in a situation where the common property allows the reporter signal peptide to be distinguished and / or separated from molecules lacking the common property. Associating one or more detectors with one or more target samples;
Each detector comprises a specific binding molecule, a carrier, and a group of blocks, the group of blocks comprising a block, the block comprising a set of reporter signals and one or more indicator signals;
And a step of detecting a block group. Associating one or more detectors with one or more target samples;
Each detector comprises a specific binding molecule, a carrier, and a group of blocks, the group of blocks comprising a block, the block comprising two or more sets of reporter signals;
A method for detecting a specimen, comprising: detecting a block group.

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